Immune modulation via C-type lectin

ABSTRACT

The invention relates to the regulation of the immune system, and in particular to the finding that the CLEC9a molecule is a marker for dendritic cells which are capable of cross-presenting extracellular antigens via the MHC class I pathway. This makes them particularly suitable for generation of cytotoxic T lymphocyte responses. Materials and methods are provided both for the induction of immune responses against target antigens, and for the inhibition or suppression of undesirable immune responses in which these cells are involved.

The present application is a §371 application of PCT/GB2008/002504 filed21 Jul. 2008 which claims priority to U.S. Provisional Application60/929,999 filed Jul. 20, 2007 and GB Application No. 0805159.1 filed 19Mar. 2008, the entire disclosures of each being incorporated byreference herein.

FIELD OF THE INVENTION

The invention relates to the regulation of the immune system, and inparticular to the use of molecules having affinity for the CLEC9amolecule to prime and inhibit immune responses to target antigens.

BACKGROUND TO THE INVENTION

The immune system is able to detect the presence of infectious agents,and trigger a response against them, without destroying self tissues.This phenomenon is not trivial, given the enormous molecular diversityof pathogens, and their high replication and mutation rates.Multi-cellular organisms have been challenged over the course ofevolution to develop several distinct immune-recognition systems, namelythe ‘innate’ and ‘adaptive’ immune systems.

The evolutionarily ancient innate immune system detects the presence andnature of infection, provides the first line of host defense, andcontrols the initiation and determination of the effector class of theadaptive immune system.

Dendritic cells (DC) play an essential role in linking innate immunityand antigen-specific adaptive responses. To initiate an immune response,DC are primed (inter alia) by pathogen-associated molecular patterns(PAMP) expressed by pathogens. Then DC orchestrate development of theadaptive immune response, much more specialized and driven byantigen-specific T- and B-cells.

The antigen recognition and uptake functions of DC against pathogens aremediated by pattern recognition receptors (PRR) that discriminate amongthe PAMPs. These PRR expressed by DC include the Toll-like receptors(TLR)¹. In addition, DC express another class of receptors, the C-typelectins, some of which may function as PRRs²⁻⁴, and/or mediateintercellular communication⁵⁻⁸. Within the C-type lectins, there is agroup of type II proteins with a single extracellular C-type lectindomain (CTLD) that are structurally and evolutionary closely related,and clustered in the NK gene complex (NKC). Although these receptorslack a calcium binding site and a typical carbohydrate recognitiondomain, they may still be able to bind carbohydrates, as shown forDectin-1⁹. Some of these receptors (CD94/NKG2, NKG2D) interact withMHC-I or related molecules and either inhibit or activate NK and T cellcytotoxicity as a result of the balance between inhibitory andactivating signals. However, for most of NK lectin receptors theirbinding specificity and relevance in NK or DC function is not known.Thus, C type lectins expressed on DC may act to recognise microbes, butmay also regulate the communication of DC with other cells byrecognizing specific cellular counterstructures.

The ontogeny and/or microenvironment in which DC are positioned mayresult in the expression of distinct combinations of surface receptorsby DC. For example, phenotypic criteria alone allow the classificationof mouse lymph node DCs into six main subpopulations¹⁰. Of these,conventional non-plasmacytoid DC in lymphoid tissues are traditionallysub-divided into CD8α⁻ and CD8α⁺ subpopulations. It has been argued thatdifferent DC subsets may be involved in specific recognition of certainpathogens and/or regulate different immune responses, e.g. Th1 or Th2(immunity) or regulatory T cells (tolerance)¹¹. However, the phenotypeand functional behavior of DCs is also significantly conditioned byexternal activating stimuli, denoting significant plasticity. As a firstapproach to understanding the differences between DC subsets, DCsubpopulations were isolated and their properties in vitro wereassessed: in mouse, CD8α⁺ and CD8α⁻ subsets of spleen DC differ in theirability to make IL-12 in vitro^(12,13). However, the differential IL-12production in vitro was also determined by the pattern recognition,demonstrating functional flexibility of different DC subsets¹⁴. As asecond approach, DC subsets were isolated, antigen-pulsed, and thenre-infused in vivo. CD8α⁺ and CD8α⁻ subsets differentially primed Th1and Th2 responses in vivo^(15,16). Immune therapy is feasible if we candetermine molecules that are selectively expressed in a particular DCsubset. These molecules can then be targeted to alter the function ofthis subset of DCs.

SUMMARY OF THE INVENTION

The inventors have found that CLEC9a is preferentially expressed on thesubset of dendritic cells that express CD8 in mice, and so aredesignated CD8⁺ dendritic cells. This is an important cell type becauseit is believed to be capable of processing antigens derived from outsidethe cell and presenting them to T cells via MHC class I molecules. Thisis in contrast to most antigen presenting cells, which presentextracellularly-derived antigens via MHC class II molecules.Consequently, this mechanism of antigen presentation is sometimesreferred to as “cross-presentation”. These cells therefore play animportant role in the generation and stimulation of cytotoxic T cell(CTL) responses, which are an essential part of the immune responseagainst intracellular pathogens (e.g. viruses) and cancers.

This finding opens up a number of applications. For example, it enablesantigens to be specifically targeted to dendritic cells capable ofcross-presentation.

Thus, in a first aspect, the invention provides a method for targetingan antigen to an antigen presenting cell, comprising contacting theantigen presenting cell with a composition comprising the antigen,wherein the antigen is associated with a binding agent having affinityfor CLEC9a, and wherein the antigen presenting cell expresses CLEC9a.

The method may be applied in vitro or in vivo. The antigen presentingcell will typically be a dendritic cell, and preferably is capable ofcross-presenting extracellular antigen via MHC class I molecules. By“extracellular” is meant that the antigen has been taken up by the cellfrom its extracellular environment, typically by endocytosis orphagocytosis.

The invention further provides a method for stimulating an immuneresponse against an antigen in a subject, comprising administering tothe subject a composition comprising the antigen, wherein the antigen isassociated with a binding agent having affinity for CLEC9a.

The method may comprise a single administration, or a sequence of two ormore administrations separated by suitably determined intervals of time.For example, the method may comprise a priming step (i.e. a firstadministration) followed by one or more boosting steps (a subsequentadministration or administrations). For example, a first administrationand second administration may be separated by one or more days, one ormore weeks, or one or more months, preferably between two weeks and onemonth. Subsequent administrations may be provided after one or moreweeks or months.

Immune responses stimulated via CLEC9a targeting involve proliferationof T cells, which may be CTL or helper T cells. Antigen presenting cells(and in particular dendritic cells) expressing CLEC9a can induceproliferation of both CD8+ T cells and CD4+ T cells, and may stimulateproliferation of both types of T cell in any given immune response.

Under certain conditions, it is believed that they are capable ofstimulating regulatory T cell (Treg) proliferation. Treg cells arecharacterised by the expression of the Foxp3 (Forkhead box p3)transcription factor. Most Treg cells are CD4+ and CD25+, and can beregarded as a subset of helper T cells, although a small population maybe CD8+. Thus the immune response which is to be stimulated by a methodof the invention may comprise inducing proliferation of Treg cells inresponse to an antigen. Thus the method may comprise administering tothe subject a composition comprising the antigen, wherein the antigen isassociated with a binding agent having affinity for CLEC9a. The antigenmay be administered with an adjuvant which promotes proliferation ofTreg cells.

Insofar as this method involves stimulating proliferation anddifferentiation of Treg cells in response to a specific antigen, it canbe considered to be a method of stimulating an immune response. However,given that Treg cells may be capable of modulating the response of othercells of the immune system against an antigen in other ways, e.g.inhibiting or suppressing their activity, the effect on the immunesystem as a whole may be to modulate (e.g. suppress or inhibit) theresponse against that antigen. Thus the methods of this aspect of theinvention can equally be referred to as methods of modulating (e.g.inhibiting or suppressing) an immune response against an antigen.

In practice, then, these methods of the invention may be usedtherapeutically or prophylactically to inhibit or suppress anundesirable immune response against a particular antigen, even in asubject with pre-existing immunity or an on-going immune response tothat antigen. This may be particularly useful (for example) in thetreatment of autoimmune disease.

Under certain conditions, it may also be possible to tolerise a subjectagainst a particular antigen by targeting the antigen to an antigenpresenting cell expressing CLEC9a. The invention thus provides a methodfor inducing tolerance in a subject towards an antigen, comprisingadministering to the subject a composition comprising the antigen,wherein the antigen is associated with a binding agent having affinityfor CLEC9a, and wherein the antigen is administered in the absence of anadjuvant.

Tolerance in this context typically involves depletion of immune cellswhich would otherwise be capable of responding to that antigen, orinducing a lasting reduction in responsiveness to an antigen in suchimmune cells.

Typically the subject is vertebrate, preferably a mammal. The subjectmay be a human, other primate, or a domestic, laboratory or livestockanimal, such as a mouse, rat, guinea pig, lagomorph (e.g. rabbit), cat,dog, pig, cow, horse, sheep or goat.

The invention further provides a composition comprising an antigen,wherein the antigen is associated with a binding agent having affinityfor CLEC9a. The composition may be a pharmaceutical composition, e.g. avaccine, containing the antigen and its associated binding agent incombination with a pharmaceutically acceptable carrier. It may beformulated for any suitable route of administration, including but notlimited to intravenous, intramuscular, intraperitoneal, nasal,subcutaneous, intradermal, etc.

The invention further provides a composition comprising an antigen foruse in a method of medical treatment, wherein the antigen is associatedwith a binding agent having affinity for CLEC9a.

Also provided is a composition comprising an antigen, for use instimulating an immune response against the antigen, wherein the antigenis associated with a binding agent having affinity for CLEC9a.

Also provided is the use of a composition comprising an antigen in thepreparation of a medicament for stimulating an immune response againstthe antigen, wherein the antigen is associated with a binding agenthaving affinity for CLEC9a. CD8α⁺ dendritic cells may be implicated inat least Th1, Th2, and Th17-type immune responses. Thus the methods ofthe invention may be applied to stimulation of various types of immuneresponse against any antigen. However these cells are believed to beparticularly important in the generation of CTL responses, so the immuneresponse to be stimulated is preferably a CTL response. The method maycomprise determining production and/or proliferation of CTLs, which aretypically T cells expressing CD8 and are capable of cytotoxic activityagainst cells displaying their cognate antigen in the context of MHCclass I molecules.

Nevertheless, targeting of antigen to CLEC9a+ dendritic cells can resultin proliferation of helper T cells as well as, or instead of, CTLs. Thusthe method may additionally or alternatively comprise determiningproduction and/or proliferation of helper T cells. The helper T cellsmay be CD4+ T cells, and may be of Th1, Th2, Th17 or Treg type. They mayalso include other types of Treg cells which do not express CD4, e.g.CD8+ Treg cells.

It will therefore be understood that the methods and compositionsdescribed above may be used for the prophylaxis and/or treatment of anycondition in which it is desirable to induce a CTL response, such ascancer, or infection by an intracellular parasite or pathogen, such as aviral infection.

It may be desirable also to administer further immunostimulatory agentsin order to achieve maximal CTL stimulation and proliferation, and/orstimulation and proliferation of other T cell types. These may includeagents capable of activating dendritic cells and stimulating theirability to promote T cell activation. Such an agent may be referred toas an adjuvant. The adjuvant may comprise an agonist for CD40 (such assoluble CD40 ligand, or an agonist antibody specific for CD40), anagonist of CD28, CD27 or OX40 (e.g. an agonist antibody specific for oneof those molecules), a CTLA-4 antagonist (e.g. a blocking antibodyspecific for CTlA-4), and/or a Toll-like receptor (TLR) agonist, and/orany other agent capable of inducing dendritic cell activation. A TLRagonist is a substance which activates a Toll-like receptor. Preferably,the TLR agonist is an activator of TLR3, TLR4, TLR5, TLR7 or TLR9. Asuitable TLR agonist is MPL (monophosphoryl lipid A), which binds TLR4.Other TLR agonists which may be used are LTA (lipoteichoic acid, whichbinds TLR2; Poly I:C (polyinosine-polycytidylic acid), which binds TLR3;flagellin, which binds TLR5; imiquimod or polyU RNA(1-(2-methylpropyl)-1H-imidazo(4,5-c)quinolin-4-amine), which binds TLR7and CpG (DNA CpG motifs), which binds TLR9; or any other component whichbinds to and activates a TLR. For more details, see Reis e Sousa,Toll-like receptors and dendritic cells. Seminars in Immunology 16:27,2004. Adjuvants which may not work via TLRs include 5′ triphosphate RNA,poly I:C, and β-glucans such as curdlan (β-1,3-glucan). Pro-inflammatorycytokines such as TNF-α or IL-1 may also be used as adjuvants.

Binding agents which have CLEC9a agonist activity (e.g. are capable ofcross-linking CLEC9a on the surface of dendritic cells, discussed inmore detail below) may also be capable of activating dendritic cells.Such agonist binding agents may therefore be considered adjuvants intheir own right. Thus, when such a binding agent is used, it may not benecessary to administer a further adjuvant such as those described above(although it may still be desirable to do so). Binding agents capable ofacting as CLEC9a agonists are discussed in more detail below.

Without wishing to be bound by theory, it is believed that the nature ofthe adjuvant used may affect the type of response obtained. Antigenpresenting cells expressing CLEC9a can stimulate both CD4+ T cells andCD8+ T cells, and the nature of the CD4+ response in particular may beaffected by the adjuvant used. For example, use of poly I:C appears tofavour generation of a Th1-type CD4+ response. Curdlan appears tostimulate a Th17-type CD4+ response

Certain adjuvants promote stimulation of Treg cells. These includeretinoic acid, and in particular all-trans retinoic acid (ATRA), alsoknown as trenitoin. Thus, when the immune response to be stimulated is aTreg response (which may in practice suppress responses of othercomponents of the immune system against a particular antigen) it may beappropriate to use a Treg-promoting adjuvant. It may also be possible tostimulate Treg cell stimulation without administration of an adjuvant.

The compositions of the invention may be administered with or formulatedfor administration with the adjuvant, either sequentially orsimultaneously, in the same or separate compositions. Thus thecompositions of the invention may, but need not, comprise an adjuvant.

Without wishing to be bound by theory, and as explained above, it isbelieved that administration of the antigen in the absence of anadjuvant may result in the development of tolerance to the antigen. Thatis to say, the immune system is induced not to respond to futureadministrations of the same antigen. This may (but need not) involve thegeneration of Treg cells which are capable of active suppression of theresponse. Thus further administrations of an antigen to a subject whohas been tolerised to that antigen should result in a lesser immuneresponse than in a subject who is naïve for that antigen (i.e. whoseimmune system has not previously been exposed to the antigen). Themagnitude of the immune response may be assessed by any appropriatecriteria, such as appearance of inflammation, swelling, cellproliferation (e.g. of Th1, Th2 or Th17 CD4+ T cells, or CTLs) orinflammatory cytokine production (e.g. IL-1, IL-4, IL-12, IFN-gamma,TNF-alpha). In certain embodiments, the tolerised individual willdisplay substantially no immune response to that antigen.

In the above-described compositions and methods, the antigen isphysically associated with the binding agent, which may be via covalentor non-covalent (e.g. electrostatic or van der Waals) interactions.Preferably the antigen is covalently coupled to the binding agent. Forexample, the binding agent may be coupled to the antigen via a suitablecoupling reagent. The skilled person is well aware of suitable methodsand reagents which may be used for such coupling reactions.

Alternatively, the antigen and binding agent may be part of the samepeptide chain, i.e. they may be expressed as a fusion protein. Thefusion protein may contain a linker sequence between the antigen andbinding agent. Alternatively, the antigen and binding agent may be inphysical proximity, e.g., a liposome, without chemical linkage.

The binding agent may be any suitable molecule having a sufficientlyspecific affinity for CLEC9a. Wherever such a binding agent is referredto throughout this specification, it may be a protein, nucleic acid(e.g. an aptamer), carbohydrate, or a small molecule. It may be aphysiological ligand for CLEC9a or a variant or analogue thereof.However, antibodies against CLEC9a and functional fragments thereof areparticularly preferred. Thus the binding agent may comprise an antibodybinding site specific for CLEC9a. The binding agent may be polyvalent asdescribed in more detail below.

The antigen is a peptide antigen. The term “peptide” refers to thenature of the antigen, i.e. that it is formed from amino acids linked bypeptide bonds, and should not be taken to imply any particular size orlength. Typically the peptide antigen will be at least 8 amino acids inlength, and may be up to 30 amino acids in length, up to 50 amino acidsin length, up to 100 amino acids, up to 200 amino acids, or even longerand may have residues coupled to the amino acids, such as glycon chains.For example, it may be 25 to 35 amino acids in length.

Without wishing to be bound by any particular theory, the peptideantigen should be capable of binding to a MHC class II or MHC Class Imolecule, or should be capable of being processed within anantigen-presenting cell (such as a dendritic cell) to give rise to oneor more peptides capable of binding to a MHC class II molecule or MHCClass I. It has recently been suggested that short epitope peptides ofaround 8 amino acids in length may induce less sustained CTL reactivitythan longer peptides (e.g. around 30 amino acids in length) (21). MHCclass I molecules typically bind peptides of 8 or 9 amino acids inlength, while MHC class II molecules can bind peptides from 8 aminoacids up to 20 amino acids, up 30 amino acids, or even longer.

The antigen may be any protein or fragment thereof against which it isdesirable to raise an immune response, in particular a CTL response, butalso a Th17 response or a Treg response. These may include antigensassociated with, expressed by, displayed on, or secreted by cellsagainst which it is desirable to stimulate a CTL response, includingcancer cells and cells containing intracellular pathogens or parasites.For example, the antigen may be, or may comprise, an epitope peptidefrom a protein expressed by an intracellular pathogen or parasite (suchas a viral protein) or from a protein expressed by a cancer or tumourcell. Thus the antigen may be a tumour-specific antigen. The term“tumour-specific” antigen should not be interpreted as being restrictedto antigens from solid tumours, but to encompass antigens expressedspecifically by any cancerous, transformed or malignant cell.

It may be particularly desirable to raise a Treg response against anantigen to which the subject exhibits, or is at risk of developing, anundesirable immune response. For example, it may be a self antigenagainst which an immune response occurs in an autoimmune disease.Examples of autoimmune diseases in which specific antigens have beenidentified as potentially pathogenically significant include multiplesclerosis (myelin basic protein), insulin-dependent diabetes mellitus(glutamic acid decarboxylase), insulin-resistant diabetes mellitus(insulin receptor), coeliac disease (gliadin), bullous pemphigoid(collagen type XVII), auto-immune haemolytic anaemia (Rh protein),auto-immune thrombocytopenia (GpIIb/IIIa), myaesthenia gravis(acetylcholine receptor), Graves' disease (thyroid-stimulating hormonereceptor), glomerulonephritis, such as Goodpasture's disease(alpha3(IV)NC1 collagen), and pernicious anaemia (intrinsic factor).Alternatively the target antigen may be an exogenous antigen whichstimulates a response which also causes damage to host tissues. Forexample, acute rheumatic fever is caused by an antibody response to aStreptococcal antigen which cross-reacts with a cardiac muscle cellantigen. Thus these antigens, or particular fragments or epitopesthereof may be suitable antigens for use in the present invention.

Depletion of Treg cells or impairment of Treg cell function has beenshown to result in autoimmune disease in murine models. Disease causedin test animals include arthritis (e.g. rheumatoid arthritis),inflammatory bowel disease, gastritis, pernicious anaemia, thyroiditis,insulitis, diabetes, sialoadenitis, adrenalitis, autoimmuneorchitis/oophoritis, glomerulonephritis, chronic obstructive pulmonarydisease and experimental autoimmune encephalitis and multiple sclerosis.

Induction of a regulatory T cell type 1 response has also been shown toreduce the development of atherosclerosis in murine models (Mallat Z. etal. Circulation 108:1232-7, 2003).

Treg activity has also been shown to be significant in the rate at whichallografts are rejected. Depletion of Treg cells or impairment offunction accelerates the rate of rejection, while infusion of testanimals with syngeneic lymphocytes enriched in Treg cells has been shownto prolong graft survival.

The methods of the present invention may therefore find use in thetreatment of any of these conditions.

It may also be possible to stimulate an immune response (whether a CTLresponse or any other kind of T cell response, including a Tregresponse) against an antigen which is present within the subject's bodywithout directly administering the antigen itself to the subject.

This may be achieved using a binding agent having affinity for theantigen and also having affinity for CLEC9a, such that the binding agentis capable of forming a complex with the antigen within the subject'sbody and targeting the antigen to an antigen presenting cell expressingCLEC9a.

The invention therefore provides a binding agent having a first bindingsite having affinity for CLEC9 and a second binding site having affinityfor the antigen. Such binding agents will be referred to in thisspecification as “bispecific” binding agents. However it will beunderstood that the binding agents may have further binding sites withalternative binding affinities, e.g. for other antigens, and the term“bispecific” should be construed accordingly.

The invention further provides a bispecific binding agent as describedabove for use in a method of medical treatment, for example, in thestimulation of an immune response against a target cell. The target cellmay be a cancer cell or a parasitised cell.

Further provided is use of a bispecific binding agent as described abovein the manufacture of a medicament for the stimulation of an immuneresponse against a target cell. The target cell may be a cancer cell ora parasitised cell.

Further provided is a method of stimulating an immune response against atarget cell in a subject, comprising administering a bispecific bindingagent to said subject. The subject may (for example) be suffering fromcancer or from an intracellular parasitic infection.

The bispecific binding agent may be formulated for administration inconjunction with an adjuvant as described elsewhere in thisspecification, or may be formulated in the same composition as anadjuvant. The nature of the adjuvant may be selected depending on thenature of the desired response. Thus, for example, ATRA may be usedwhere it is desirable to induce a Treg response against the antigen,curdlan may be used where a Th17 response is desired, and any othersuitable adjuvant (e.g. anti-CD40, poly I:C, etc.) may be used wheremore conventional CTL responses are required. Where no adjuvant isadministered, it may be possible to induce stimulation of Treg cellsand/or tolerance to the antigen.

The invention further provides a pharmaceutical composition comprising abispecific binding agent as described above, in admixture with apharmaceutically acceptable carrier. The composition may furthercomprise an adjuvant, or may be for administration in conjunction withan adjuvant, as described elsewhere in this specification.

In some embodiments either or both binding sites may be antibody bindingsites specific for CLEC9a or the antigen respectively. Thus the bindingagent may be a bispecific antibody comprising at least a first antibodybinding site specific for CLEC9a and at least a second antibody bindingsite having affinity for the antigen. The term “bispecific antibody”should be interpreted to encompass any molecule or molecular complexhaving such two binding sites, such as bispecific single chain Fv dimersand “diabodies” (see below).

The finding that the CLEC9a molecule is a marker for a specific subsetof dendritic cells also makes it a suitable therapeutic target fordownregulation of undesirable immune responses.

Thus in a further aspect, the invention provides a method of inhibitingan immune response in a subject comprising administering a binding agenthaving affinity for'CLEC9a.

In this aspect of the invention, the binding agent is capable ofdirectly or indirectly inhibiting dendritic cell function in a dendriticcell to which it is bound. The binding agent may inhibit CLEC9a frombinding to its cognate ligand, or may inhibit an aspect of cell functionsuch as maturation in response to adjuvant, or antigen presentation.Alternatively, the binding agent may be directly or indirectly capableof killing the cell, which may be regarded as a “depleting” activity.Typically the binding agent comprises an “effector” moiety responsiblefor directly or indirectly killing the cell or otherwise affectingantigen presentation.

For example, the binding agent may be capable of recruiting componentsof the subject's immune system, thus stimulating an immune attack on thecell. For example, an antibody Fc region may be used to recruitcomponents of the complement system, which may lead to lysis of the cellvia the lytic pathway of complement, or opsonisation of the cell andsubsequent phagocytosis by phagocytic cells of the immune system such asneutrophils or macrophages. Phagocytes also possess Fc receptors whichenable them to phagocytose cells bound by antibodies; thus antibodiescan themselves mark cells for phagocytosis without activation ofcomplement. Thus the effector moiety may comprise an antibody Fc region,for example of IgG or IgM.

The binding agent may comprise other types of effector moiety which actto kill targeted cells. For example, it may comprise a toxin moleculecapable of killing the cell. This mechanism may be particularlyeffective, as the inventors have shown that antibodies binding CLEC9acan be endocytosed by dendritic cells, so a conjugated toxin moleculewould have a high probability of being taken up into the cell.

Alternatively the effector moiety may be an enzyme capable of activatinga prodrug in the vicinity of the cell, for example converting anon-toxic molecule into a toxic molecule.

As already described, the binding agent may comprise an antibody bindingsite specific for CLEC9a.

Useful binding agents therefore include molecules comprising an antibodybinding site specific for CLEC9a, and one or more of an antibody Fcregion, a toxin molecule, or an enzyme capable of activating a prodrug.

The binding agent may itself be a functional antagonist of CLEC9a. Thus,it may simply be sufficient for the binding agent to bind to CLEC9a inorder to exert its inhibitory function, by preventing CLEC9a frombinding to its cognate ligand, or from exerting its normal signallingrole, e.g. in dendritic cell maturation and/or antigen presentation. Insuch embodiments, it may be desirable that the binding agent comprisesonly one or two binding sites (e.g. antibody binding sites) specific forCLEC9a. For example, the binding agent may be (or comprise) an antibodyor an antibody fragment, such as a Fab or scFv fragment.

Antagonists which prevent binding of CLEC9a to its ligand may beparticularly useful in the treatment of autoimmune diseases. Someautoimmune diseases are characterised by unusually high levels of celldeath and it is believed that immune responses against self antigensassociated with these cells may contribute to the pathogenesis of theseconditions. CLEC9a antagonists may therefore be used to prevent CLEC9afrom binding to the ligand exposed in dead and dying cells (especiallythose undergoing immunogenic cell death) and may thus inhibit or preventstimulation of immune responses against these antigens.

Other functional antagonists of CLEC9a may also be useful in the methodsdescribed. Such antagonists include nucleic acids or analogues thereofcapable of hybridising to mRNA or DNA encoding CLEC9a (for example,ribozymes, antisense RNA or DNA molecules, siRNA, etc.), small moleculeantagonists of CLEC9a, etc.

Yet further CLEC9a antagonists are competitors for the CLEC9a ligand,which can block binding sites on the ligand for dendriticcell-associated CLEC9a and so prevent the ligand from being recognisedor bound by the dendritic cell. Suitable competitors include solublemolecules comprising the extracellular domain of CLEC9a or a portionthereof sufficient to bind to the CLEC9a ligand.

The CLEC9a extracellular domain (or portion thereof) may be associatedwith a heterologous moiety which may modulate some property of theantagonist, such as its pharmacokinetic properties in vivo. Theextracellular domain may be covalently or non-covalently bound to theheterologous moiety, or may be expressed as a fusion protein with theheterologous moiety. For example, the heterologous moiety may be anantibody Fc domain, in order to provide increased serum half life andallow efficient clearance of complexes between the antagonist and theCLEC9a ligand.

Other possible functions of the heterologous moiety include mediatingoligomerisation of the CLEC9a extracellular domain, and facilitationpurification of the antagonist or isolation from a sample. For example,a suitable antagonist may be a soluble molecule comprising or consistingof the CLEC9a extracellular domain (or a fragment thereof sufficient tobind CLEC9a ligand) associated with an avidin monomer. The avidinmonomers will tend to associate into tetramers, providing a complexcomprising four CLEC9a domains and four avidin subunits. This constructcan readily be isolated by contact with biotin, which may be provided ona solid support such as a bead.

Where the binding agent or antagonist is a protein, it may be possibleto administer a nucleic acid (e.g. DNA) encoding the antagonist.Typically the nucleic acid will be taken up by cells within the body(e.g. muscle cells), expressed, and secreted from those cells. Thisapproach is often referred to as DNA vaccination.

Antibodies are particularly suitable as binding agents and antagonists,and can conveniently be expressed in scFv form. If necessary, anantibody can be encoded as a fusion protein with the antigen, or with aneffector moiety as described above. An example of a DNA vaccinationapproach is described in Nchinda et al., J. Clin. Invest. 118(4),1427-36, 2008.

The nucleic acid typically comprises a coding region encoding thebinding agent or antagonist, optionally in conjunction with any desiredfusion partner, in operable linkage with transcriptional andtranslational regulatory sequences to ensure appropriate expression andsecretion of the protein from cells which take up the nucleic acid. Suchsequences include (but need not be limited to) transcriptionalinitiation sequences (e.g. promoter and enhancer), transcriptionaltermination sequences, appropriate splicing signals, translationalinitiation and termination sequences, and a signal peptide to enablesecretion.

Thus the invention further provides a nucleic acid (e.g. a DNA) encodinga CLEC9a antagonist or binding agent, for use in a method of medicaltreatment. Also provided is a nucleic acid encoding a CLEC9a antagonistor binding agent for use in a method of and therapeutic uses thereof.

The subject to whom the binding agent or antagonist is administered maybe suffering from an inflammatory or autoimmune condition, especially acondition characterised by undesirable CTL activity, and/or a conditioncharacterised by high levels of cell death. Such conditions include:

-   -   autoimmune diseases, including rheumatoid arthritis and other        types of chronic or acute arthritis or arthropathies with an        immune component, systemic lupus erythematosus (which is known        to involve particularly high levels of cell death), scleroderma,        Sjögren syndrome, autoimmune (particularly Type I) diabetes,        thyroiditis, and other organ-specific immune diseases, including        psoriasis;    -   neurologic diseases, including multiple sclerosis, myasthenia        gravis, and other neurologic immune-mediated diseases. Also        included are gastrointestinal diseases, including Crohn's        disease, colitis, celiac disease and hepatitis;    -   cardiovascular diseases, which are now recognised to have a        significant immune-mediated component, including        atherosclerosis, cardiomyopathy, rheumatic fever, endocarditis,        vasculitis, and other immune-mediated cardiovascular diseases;    -   immune-mediated respiratory diseases, including emphysema,        respiratory airways infections, and other immune-mediated        respiratory diseases;    -   allergic processes and hypersensitivity reactions (type I, II,        III, and IV), including asthma, rhinitis, and other        immune-mediated hypersensitivity reactions;    -   transplant or graft rejection and graft versus host disease, as        occurs during or subsequent to, for example, organ transplant,        tissue graft, blood transfusion, bone marrow transplant;    -   immunopathological responses to infectious agents, including        septic shock syndromes;    -   degenerative processes, such as neurodegenerative processes,        that implicate immune competent cells such as microglia.

The invention further provides a binding agent having affinity forCLEC9a, or a CLEC9a antagonist, for use in a method of medicaltreatment.

Also provided is a binding agent having affinity for CLEC9a, or a CLEC9aantagonist, for use in the inhibition of an immune response.

Also provided is the use of a binding agent having affinity for CLEC9a,or a CLEC9a antagonist, in the preparation of a medicament for theinhibition of an immune response.

The inventors have also found that CLEC9a agonists are capable ofactivating dendritic cells, and may therefore be useful in stimulatingimmune function, even when not physically associated with an antigen.Thus the invention provides a method of stimulating an immune responsein a subject, comprising administering a binding agent having affinityfor CLEC9a.

The CLEC9a agonist may be a binding agent having affinity for CLEC9a,and may comprise an antibody binding site specific for CLEC9a.

Without wishing to be bound by theory, it is believed that bindingagents having more than two binding sites (e.g. antibody binding sites)specific for CLEC9a may be particularly effective agonists of CLEC9aactivity, probably because they can cross-link or cause association ormultimerisation of CLEC9a on a cell surface. Binding agents may bereferred to as “bivalent” if they possess two such binding sites or“polyvalent” if they possess more than two such binding sites. Thereforethe binding agent is preferably polyvalent, and may comprise at leastthree, four, five, ten or even more binding sites. Such binding agentsmay comprise a plurality of binding sites immobilised in or on aparticle, such as a bead (e.g. of latex), a liposome or vesicle, or anyother suitable particle. Thus the binding sites may be immobilised on orin a particulate solid phase. Alternatively a polyvalent binding agentmay simply comprise more than two binding sites covalently linked orotherwise associated with one another. For example, whole antibodies orfunctional fragments thereof (see below) may be associated as fusionproteins and/or by chemical cross-linking. The skilled person is wellaware of suitable techniques for preparing such polyvalent bindingagents. When the methods are performed in vitro, binding agents (such asantibodies against Clec9a) immobilised on a surface of the culturevessel can be used as agonists; in this case the coated surface of theculture vessel may itself be considered a polyvalent binding agent.

The agonist may be administered alone, in order to stimulate immunefunction generally, or in conjunction with a target antigen againstwhich it is desirable to induce an immune response. The agonist andantigen can be administered separately or in the same composition,simultaneously or sequentially, as desired. The agonist may bephysically associated with the antigen as described above in relation tothe first aspect of the invention, or the two components may bephysically separate, distinct entities.

As already described, the methods and compositions described may beparticularly useful for the prophylaxis and/or treatment of anycondition in which it is desirable to induce a CTL response, such ascancer, or infection by an intracellular parasite or pathogen, such as aviral infection.

The methods and compositions described may also used for the prophylaxisand/or treatment of any condition in which it is desirable to induce aTreg response, e.g. a condition involving an undesirable orinappropriate immune response against a particular condition, such as anautoimmune disease.

A further immunostimulatory agent or adjuvant as described above mayalso be administered in association with the agonist and optionally theantigen. Thus the adjuvant may comprise, for example, a CD40 agonist ora TLR agonist. The nature of the adjuvant may be selected depending onthe nature of the desired immune response. Thus, for example, if aTh17-type CD4 T cell response or a Treg response is desirable, theadjuvant may be selected accordingly.

Identification of CLEC9a as a marker for a subset of dendritic cellsprovides means for identification and isolation of such cells frombiological samples.

Thus, in a further aspect, the invention provides a method of detectingan antigen presenting cell in a sample, comprising contacting the samplewith a binding agent having affinity for CLEC9a and determining bindingof the binding agent to one or more cells.

The method also provides a method of isolating an antigen presentingcell from a sample, comprising contacting the sample with a bindingagent having affinity for CLEC9a and isolating one or more cells towhich the binding agent is bound. The binding agent may be immobilisedon a solid support (such as a magnetic bead) in order to facilitateisolation.

As will be clear from the discussion above, the antigen presenting cellis typically a dendritic cell, and may be capable of cross-presentingextra-cellular antigen via MHC class I molecules.

In order to confirm that the cells identified by the binding agent forCLEC9a are dendritic cells, or to enrich a sample for dendritic cellsbefore contacting the cells with the binding agent for CLEC9a, themethod may comprise the step of contacting the sample with a secondbinding agent having affinity for a dendritic cell marker anddetermining binding of the second binding agent to one or more cells.The two binding agents may be contacted with the cells simultaneously orsequentially, and in any order. In some embodiments, only those cells towhich both the first and second binding agents bind are identified orisolated.

The dendritic cell marker may be a pan-dendritic cell marker such asCD11, especially CD11c (in mice).

For samples of human dendritic cells, the dendritic cell marker may beHLA-DR. It may be desirable to confirm that the cells arelineage-negative, i.e. they do not express CD3, CD14, CD19 or CD56.

For samples of human cells, it may be desirable to confirm that thecells identified by the binding agent for CLEC9a also express BDCA-3(also known as CD141 or thrombomodulin). The method may thereforecomprise the step of contacting the sample with a further binding agenthaving affinity for BDCA-3 and determining binding of the furtherbinding agent to one or more cells, and/or isolating cells to which thefurther binding agent binds. The binding agents for CLEC9a and BDCA-3may be contacted with the cells simultaneously or sequentially, and inany order. In some embodiments, only those cells to which both bindingagents bind are identified or isolated.

Additionally or alternatively, it may be desirable to enrich the samplefor the desired cell type by % negative selection for one or moreunwanted cell types. The unwanted cell types may comprise othersubgroups of dendritic cells such as plasmacytoid dendritic cells(pDCs), which may be excluded by negative selection for CD123 or Ly6C.Negative selection may be performed before, simultaneously with, orafter selection for cells expressing CLEC9a. Negative selection may beperformed for CD3, CD14, CD19, and/or CD56.

It may also be desirable to determine the level of CLEC9a expression onthe cells. This may make it possible only to select cells which expressa desired level of CLEC9a, e.g. a higher level of CLEC9a than anotherpopulation of cells which expresses CLEC9a at a detectable level. Forexample, without wishing to be bound by theory, it is believed that thesubset of DCs which expresses CD8 (or is equivalent to that subset inhumans) expresses a higher level of CLEC9a than pDCs. It may thereforebe possible to select CD8 DCs or their equivalents by only selectingthose cells which express CLEC9a above a certain threshold level.

The binding agents may be labelled to facilitate detection and/orisolation of the cells, e.g. with a label capable of emitting adetectable signal (such as a fluorescent or radioactive label) or withan affinity tag capable of being specifically bound by a bindingpartner. Examples of affinity tags and binding partners include epitopesand cognate antigens, positively charged peptides (e.g. poly-His) andmetal (e.g. nickel) ions, avidin/streptavidin and biotin, carbohydratesand lectins, etc. The skilled person will be able to design a suitablesystem depending on their specific requirements.

Identification or isolation of the cells may involve contacting thesample with one or more detecting agents capable of binding to the firstand/or second binding agent. The detecting agent may itself be labelledas described above.

To facilitate isolation or detection, the binding or detecting agent maybe immobilised on a solid support.

The invention further provides a population of antigen presenting cellsisolated by a method as described above.

The cells isolated by these methods may be used for various purposes,including in vitro study and ex vivo therapy. For example, isolatedcells may be pulsed with a desired antigen in vitro. The cells may thenbe administered to a subject in order to stimulate an immune responseagainst the antigen.

Thus, the invention further provides a method of stimulating an immuneresponse against a peptide antigen comprising providing an antigenpresenting cell or population thereof isolated by a method as describedabove, and contacting said cell or population of cells with saidantigen.

Preferably, the antigen presenting cells present said antigen or afragment thereof in the context of MHC class I molecules.

Following said contacting step, the cell or population of cells may beadministered to a subject. Preferably the cells are re-administered tothe subject from whom they were derived.

The cell or population of cells may also be contacted with an adjuvant,as described above. Contacting may take place in vitro, for example ator approximately at the same time as contacting with the antigen, or ator after administration to the recipient subject. Contact with theadjuvant may stimulate the capacity of the cell or population of cellsto activate or promote proliferation of T cells in response to theantigen. The adjuvant may be administered simultaneously with the cells,or sequentially, in the same or different compositions. Theimmunostimulatory adjuvant may be, for example, a CD40 agonist or a TLRagonist. The nature of the adjuvant may be selected depending on thenature of the desired immune response. Thus, for example, if a Th17-typeCD4 T cell response or a Treg response is desirable, the adjuvant may beselected accordingly.

The invention therefore provides a primed antigen presenting cell orpopulation thereof, obtained by the methods described above. By “primed”is meant that the cell has been contacted with an antigen, is presentingthat antigen or an epitope thereof in the context of MHC molecules,preferably MHC I molecules, and is capable of activating or stimulatingT cells to proliferate and differentiate into effector cells in responsethereto.

Also provided is a primed antigen presenting cell or population thereof,obtained by the methods described above, for use in a method of medicaltreatment, and especially for use in a method of stimulating the immuneresponse against a target antigen. Also provided is the use of a primedantigen presenting cell or population thereof, obtained by the methodsdescribed above, in the preparation of a medicament for the stimulationof an immune response against a target antigen.

Alternatively the primed cells may be contacted with T cells in vitro inorder to generate T cells (particularly CTLs, but also CD4+ T cells,including Th17 and Treg cells) specific for the antigen. Thus, followingsaid contacting step, the method may comprise contacting said antigenpresenting cells with a population of cells comprising one or more Tcells. The T cells in the population may be allowed to expand in culturein order to increase the number or proportion of T cells in thepopulation which are specific for the antigen. The T cells may then beadministered to a subject. Optionally the T cells are separated fromother cells in the population before administration.

the population of cells may also be Contacted with an adjuvant, forexample at substantially the same time as they are contacted with theprimed antigen presetting cells.

Preferably, the T cells and antigen presenting cells are autologous,i.e. they are derived from the same subject, or from geneticallyidentical subjects.

The T cells may be re-administered to the subject from whom they (ortheir progenitors) were derived.

Again, an adjuvant may be administered with the T cells, when they areadministered to the subject. The adjuvant may be a CD40 agonist (such asan antibody specific for CD40) or a TLR agonist. The adjuvant may beadministered simultaneously with the T cells or sequentially, in thesame or different compositions.

The invention therefore further provides a T cell or population thereof,obtained by the methods described above. Also provided is a T cell orpopulation thereof, obtained by the methods described above, for use ina method of medical treatment, and especially for use in a method ofstimulating an immune response against a target antigen. Also providedis the use of a T cell or population thereof, obtained by the methodsdescribed above, in the preparation of a medicament for the stimulationof an immune response against a target antigen.

The invention further provides an isolated population of human dendriticcells expressing CLEC9a. The population may contain at least 5, at least10, at least 100, at least 1000 or at least 10,000 dendritic cells.Preferably at least 50%, at least 60%, at least 70%, at least 80%, atleast 90%, or at least 95% of the dendritic cells within the populationexpress CLEC9a. For this purpose, dendritic cells are considered to belineage-negative, HLA-DR⁺ cells. Lineage-negative cells do not expressCD3, CD14, CD19 or CD56.

The isolated population may be comprised within a sample comprisingother cell types, as long as the required proportion of dendritic cellswithin the sample expresses CLEC9a. Thus the sample may compriselymphocytes (for example T cells, and especially CTLs), or other typesof antigen presenting cells which are not dendritic cells.

The dendritic cells expressing CLEC9a will typically be BDCA-3+. Theymay also be CD123⁻, CD34⁻, CD16⁻ and CD11b/c⁻.

The invention further provides a method of stimulating an immuneresponse against a peptide antigen comprising providing a population ofhuman dendritic cells expressing CLEC9a, and contacting said populationwith said antigen.

Following said contacting step, the population of human dendritic cellsexpressing CLEC9a may be administered to an autologous subject; i.e.they are administered to a subject from whom they were originallyderived.

The population of human dendritic cells expressing CLEC9a may also becontacted with an adjuvant, as described above. Contacting may takeplace in vitro, for example at or approximately at the same time ascontacting with the antigen, or at or after administration to therecipient subject. Contact with the adjuvant may stimulate the capacityof the population of cells to activate or promote proliferation of Tcells in response to the antigen. The adjuvant may be administeredsimultaneously with the cells, or sequentially, in the same or differentcompositions. The immunostimulatory adjuvant may be, for example, a CD40agonist or a TLR agonist. The nature of the adjuvant may be selecteddepending on the nature of the desired immune response. Thus, forexample, if a Th17-type CD4 T cell response or a Treg response isdesirable, the adjuvant may be selected accordingly.

The invention therefore provides a population of human dendritic cellsexpressing CLEC9a, primed with an antigen. By “primed” is meant that thecell has been contacted with an antigen, is presenting that antigen oran epitope thereof in the context of MHC molecules, preferably MHC Imolecules, and is capable of activating or stimulating T cells toproliferate and differentiate into effector cells in response thereto.

Also provided is a primed population of human dendritic cells expressingCLEC9a for use in a method of medical treatment, and especially for usein a method of stimulating the immune response against a target antigen.Also provided is the use of a primed population of human dendritic cellsexpressing CLEC9a in the preparation of a medicament for the stimulationof an immune response against a target antigen.

Alternatively the primed cells may be contacted with T cells in vitro inorder to generate T cells (particularly CTLs, but also helper T cells orTregs) specific for the antigen. Thus, following said contacting step,the method may comprise contacting said population of human dendriticcells expressing CLEC9a with a population of cells comprising one ormore T cells. The T cells in the population may be allowed to expand inculture in order to increase the number or proportion of T cells in thepopulation which are specific for the antigen.

The T cells may then be administered to a subject. Optionally the Tcells are separated from other cells in the population beforeadministration. As in other aspects of the invention, the dendriticcells and T cells may also be contacted with an adjuvant. The nature ofthe adjuvant may be selected depending on the nature of the desiredimmune response. Thus, for example, if a CTL response, Th17-type CD4 Tcell response or a Treg response is desirable, the adjuvant may beselected accordingly.

Preferably, the T cells and dendritic cells are autologous, i.e. theyare derived from the same subject, or from genetically identicalsubjects.

The T cells may be re-administered to the subject from whom they (ortheir progenitors) were derived.

Again, an adjuvant may be administered with the T cells. The adjuvantmay be a CD40 agonist (such as an antibody specific for CD40) or a TLRagonist. The adjuvant may be administered simultaneously with the Tcells or sequentially, in the same or different compositions.

The invention therefore further provides a T cell or population thereof,obtained by the methods described above. Also provided is a T cell orpopulation thereof, obtained by the methods described above, for use ina method of medical treatment, and especially for use in a method ofstimulating an immune response against a target antigen. Also providedis the use of a T cell or population thereof, obtained by the methodsdescribed above, in the preparation of a medicament for the stimulationof an immune response against a target antigen.

In all of the above-described aspects, the antigen may be any antigenagainst which it is desirable to stimulate an immune response,particularly a CTL response, a Th17-type response or a Treg response.For example, the antigen may be an antigen expressed by an intracellularpathogen or parasite, or may be expressed by a cancer cell, as describedelsewhere in this specification. Alternatively it may be an antigenagainst which an undesirable or inappropriate response takes place (e.g.in an autoimmune disease) and against which it is desired to stimulate aTreg response.

The surprising finding that CLEC9a binds to a ligand found on or inmammalian cells, rather than infectious agents, also makes availableassays for the identification of the ligand.

Thus the invention provides a method of screening for a physiologicalligand for CLEC9a comprising contacting a target substance comprisingthe extracellular domain of CLEC9a or a portion thereof sufficient tobind CLEC9a ligand with a test substance which is a component of amammalian cell, and determining binding of the target substance to thetest substance.

Binding may indicate that the test substance is a (or the) physiologicalligand for CLEC9a.

Where binding occurs, the method may further comprise the step ofidentifying the test substance.

It is believed that the ligand for CLEC9a is constitutively expressed insome or all healthy mammalian cells but is not accessible forinteraction with CLEC9a while the cell remains healthy. Certain types ofcell damage or cell death (especially when immunogenic, such as primaryor secondary necrosis) cause the ligand to be exposed in such a way thatit becomes accessible for interaction with CLEC9a on dendritic cells.

Thus, the test substance may be an intracellular component of amammalian cell (e.g. a healthy mammalian cell, not infected by anintracellular parasite or pathogen). It may comprise or consist of aprotein, carbohydrate, lipid or nucleic acid. It may comprise more thanone of these components, for example it may be a glycosylated proteincomprising carbohydrate and lipid components, a lipid-anchored proteincomprising lipid and protein (and optionally also carbohydrate)components, or a glycolipid comprising lipid and carbohydratecomponents.

It will be understood that the test substance may not comprise theentire molecule or substance which would be present in the mammaliancell under physiological conditions, but may comprise a portion thereofwhich is sufficient to interact with CLEC9a. For example the testsubstance may comprise an isolated domain, or even a peptide (e.g. of 5to 10 amino acids, up to 20 amino acids, up to 50 amino acids, or up to100 amino acids) from a cellular protein.

Typically the test substance will be from the same mammalian species asthe CLEC9a (or portion thereof) which is present in the targetsubstance.

The method may comprise the step of contacting the target substance witha sample (e.g. a liquid sample, such as an aqueous sample) comprisingthe test substance.

The target substance may be provided in solution (e.g. in an aqueoussolution) or may be immobilised on a solid support.

The sample may comprise permeabilised mammalian cells. By“permeabilised” is meant that the plasma membrane has become permeableby diffusion to entities which would not normally be able to cross theplasma membrane (without being actively taken up by the cell). Dyes suchas propidium iodide and TO-PRO3, to which the plasma membrane is notpermeable under normal conditions, are conventionally used to testplasma membrane integrity/permeability. Thus the plasma membrane may bepermeable to such substances. For example, it may be permeable tosubstances having a molecular weight of above 500 Da, above 1 kDa, above10 kD, above 50 kDa, above 100 kDa, or even higher. The term“permeabilised” is used here to refer to cells which substantiallyretain their cellular architecture (apart from the increasedpermeability of the plasma membrane, and potentially other membraneswithin the cell) such that individual cells can still be distinguished(e.g. by microscopy). The terms “lysate”, “extract” or “homogenate” maybe used for preparations in which the architecture of the cell isdisrupted to such an extent that individual cells can no longer bedistinguished or are no longer present.

The permeabilised cells may be necrotic. Necrosis may be primary orsecondary necrosis. Primary necrosis may be induced experimentally by(for example) irradiation (e.g. with ionising radiation such as UVlight), serum deprivation, at least one freeze/thaw cycle, or bytreatment with necrosis-inducing chemicals such as anthracyclines (suchas doxorubicin and daunorubicin) and anthracenediones (such asmitoxantrone). Secondary necrosis occurs when cells induced to enterapoptosis are not phagocytosed by neighbouring cells and the plasmamembrane subsequently becomes disrupted.

Alternatively, healthy cells may be directly permeabilised with an agentwhich disrupts or forms pores in the plasma membrane. Suitablepermeabilising agents include pore-forming agents such as saponins,(e.g. beta-escin), which precipitate cholesterol, thus removing it fromthe membrane and increasing its permeability, various bacterial toxinssuch as cytolysins (e.g. streptolysin-O from Staphylococcus aureus) andalpha-toxin from Staphylococcus aureus and detergents such as TritonX-100, Brij-96, Tween, etc.

Optionally, the cells may be fixed, either before or afterpermeabilisation. Fixation before permeabilisation may be preferred toreduce the chance that the ligand will be lost from the cell (e.g. bydiffusion) following permeabilisation. Fixation can be performed with anorganic solvent such as acetone, methanol, ethanol or mixtures thereof(which generally remove lipids and dehydrates the cell, whileprecipitating the proteins on the cellular architecture) and/or with across-linking reagent such as formaldehyde (e.g. as formalin) orparaformaldehyde (which cross-link cellular components such as proteinsetc via free reactive groups present on those cellular components, suchas amino groups).

Where the sample comprises permeabilised cells, the method may comprisethe step of determining the subcellular location at which binding of thetarget substance takes place, by detection of the target substance.

Detection may be direct or indirect. For example the target substancemay comprise a label, and the method may comprise the step ofdetermining the subcellular location of the label. Alternatively themethod may comprise the further step of contacting the target substancewith a detection agent, which may be a binding agent capable of bindingto the target substance. The detection agent may itself comprise alabel. Detection may be achieved by any suitable technique, such asmicroscopy, e.g. confocal microscopy. For example, the label may befluorescent.

Alternatively, the sample may be, or may comprise, a cell lysate,extract or a subcellular fraction of a mammalian cell. For example, itmay comprise a whole cell lysate, or a subcellular fraction which is notexposed to the external environment in an intact healthy cell. Forexample, the sample may comprise an isolated cytoplasmic fraction, anisolated nuclear fraction, an isolated endoplasmic reticulum fraction,an isolated Golgi fraction, or an isolated mitochondrial fraction.“Isolated” in this context means separated from at least one othernormal component of the intact cell (such as the plasma membrane).Subcellular organelles in such fractions, such as nuclei ormitochondria, may be intact or disrupted.

Following a positive binding reaction with a first sample, the firstsample may be further fractionated to provide a second sample whichlacks one or more components present in the first sample. The method maythen be repeated. This may assist in identification of a test substancewhich binds to the target substance. This process may be repeated asoften as desired, using progressively smaller fractions.

Additionally or alternatively the method may comprise the step ofisolating a complex comprising the target substance and the testsubstance. This may be achieved by isolating a solid support (e.g. abead) to which the target substance is bound. Alternatively a bindingagent may be employed which is capable of binding to the targetsubstance. The binding agent may be immobilised on a solid support. Thetarget substance may comprise a member of a specific binding pair andthe binding agent may comprise the second member of the specific bindingpair. For example, the binding agent may be an antibody specific for thetarget substance. Alternatively, one of the binding agent and targetsubstance may comprise an avidin/streptavidin moiety while the othercomprises a biotin moiety.

The term “specific binding pair” is used to describe a pair of moleculescomprising a specific binding member (sbm) and a binding partner (bp)therefor which have particular specificity for each other and which innormal conditions bind to each other in preference to binding to othermolecules. Examples of specific binding pairs are antibodies and theircognate epitopes/antigens, ligands (such as hormones, etc.) andreceptors, avidin/streptavidin and biotin, lectins and carbohydrates,and complementary nucleotide sequences.

To facilitate isolation, a single binding agent may be polyvalent, i.e.capable of binding simultaneously to more than one target substance. Ifthe target substance is also polyvalent (i.e. capable of bindingsimultaneously to more than one binding agent of the same type) across-linked complex may be formed.

The skilled person will be well aware of suitable techniques, such asimmunoprecipitation techniques, which may be employed.

For example, the target substance may be a soluble molecule comprisingor consisting of the CLEC9a extracellular domain associated with (e.g.conjugated to or in a fusion protein with) an avidin monomer. The avidinmonomers will tend to associate into tetramers, providing a bindingagent comprising four CLEC9a domains and four avidin subunits. Thisconstruct may thus form a complex with a test substance in an assay asdescribed here, which can then be isolated by contact with biotin, whichmay be present on a solid support such as a bead.

Alternatively the test substance may be immobilised on a solid support,such as a membrane, microtitre plate, or microarray chip. The supportmay be contacted with the target substance, and the location of anybound target substance determined.

It may be desirable to test a plurality of samples (e.g. differentcellular fractions) suspected of containing a ligand for CLEC9a to seewhether any of them do in fact contain a substance capable of binding tothe target substance. Additionally or alternatively, it may be desirableto test a plurality of known substances (e.g. proteins) to see whetherany of them is capable of binding to the target substance.

Thus a single solid support may comprise only one sample or testsubstance, or it may carry a plurality of samples or test substanceseach at a defined location on the support.

Depending on the format of the assay, the method may comprise the stepof identification of the particular support on which a positive bindingreaction takes place, or of the location on a support at which apositive binding reaction takes place. This may therefore reveal thenature of the sample which contains the test substance responsible forthe positive reaction, or it may directly reveal the identity of thetest substance.

Alternatively it may be possible to isolate a complex of the testsubstance and the target substance from the solid support and carry outfurther analysts to identify the test substance.

Whatever the format of the assay, where the test substance is a protein,it may be possible to use proteomic techniques to identify the testsubstance. This will typically combine mass spectroscopy and databaseinterrogation. The test substance (or a complex of test and targetsubstances) may be subjected to digestion with one or more proteaseswith known target cleavage sequences to yield peptides with known N- orC-terminal residues (depending on the particular protease used). Theresulting peptides are then subjected to mass spectroscopy (e.g.MALDI-TOF) to determine their molecular weights. Suitable proteinsequence databases can then be interrogated to identify proteins capableof giving rise to such peptides.

Alternatively the test substance may be displayed on the surface of acell or virus. For example, a phage display technique may be used todisplay test substances or fragments thereof on the surface of abacteriophage. Alternatively a cell may be engineered to display thetest substance on its surface. Alternatively an interaction between thetest substance and target substance may take place within a cell or acell free expression system and may induce a detectable reaction such asexpression of a reporter gene. An example of such a system is the yeasttwo-hybrid system, but the skilled person will be aware of other systemswhich rely on “bait-prey” interaction between two proteins to driveexpression of a reporter gene. Such formats may be used to screen apopulation of nucleic acid molecules (e.g. a cDNA library) in order toidentify a nucleic acid molecule which encodes a substance which canbind the target substance (if present), where a protein encoded by thenucleic acid is expressed in the cell or cell-free expression system. Insuch formats, the method may comprise the step of isolating (e.g.cloning) a nucleic acid responsible for a positive result, anddetermining the identity of the protein encoded by the nucleic acid.

Where the test substance is not a protein, other analytical techniquesmay be employed.

The invention will now be described in more detail, by way of exampleand not limitation, by reference to the accompanying drawings andexamples.

DESCRIPTION OF THE DRAWINGS

FIG. 1 shows cDNA (SEQ ID NO: 1) and amino acid sequences for the humanCLEC9a protein (SEQ ID NO: 2.

FIG. 2 shows cDNA (SEQ ID NO: 3) and amino acid (SEQ ID NO: 4) sequencesfor the mouse CLEC9a protein.

FIG. 3 shows detection of mouse CLEC9a protein expressed in humanPhoenix cells. Western blot using anti-CLEC9a on full lysates fromPhoenix cells expressing CLEC9a (FNX-C9) or parental cells (FNX) inreducing (R) and non-reducing (NR) conditions.

FIG. 4 shows distribution of mouse CLEC9a transcripts in spleen DC andin vitro cultured BMDC. mRNA from subsets of spleen DC or from purifiedsubsets of GMCSF or Flt3L-BMDC were subjected to RT-PCR using CLEC9aspecific primers (upper lanes) or β-actin primers (lower lanes) asindicated under Methods.

FIG. 5 illustrates the CD3ζ-NKRP1-CLEC9a chimera generated in this work,showing the domains presents in the chimeric molecule with theextracellular domain of CLEC9a, the transmembrane domain from NKRP1 andthe cytoplasmic domain from CD3ζ (SEQ ID NO: 5).

FIG. 6 shows expression of CLEC9a in primary cells. Splenocytes werestained with biotin anti-CLEC9a (thick line, 10 mg/ml) or biotin-ratIgG1 (thin line, isotype control) followed by streptavidin-PE (1:1000)and counterstained with CD11c, CD4, CD8 or Ly6C. Histograms show thestaining for CD11c negative cells vs CD11c positive cells, which arethen further analyzed in the CD4/CD8 subsets of conventional DCs or inthe Ly6C+ subset corresponding to pDC.

FIG. 7 is a Western blot showing Syk pull-down with CLEC9a cytoplasmicpeptide. A biotinylated peptide corresponding to CLEC9a cytoplasmicdomain or a Dectin-1 control was used to pull down recombinant Syk asindicated in Methods.

FIG. 8 shows kinase activation with anti-CLEC9a. LK cells expressingCLEC9a were activated for the indicated times with plated antibodies andthen were lysed and subjected to SDS-PAGE and WB with anti-P-Syk oranti-P-Erk antibodies to detect activation of these pathways. Proteincontrols with anti-Syk or anti-Erk indicate the total kinase.

FIG. 9 shows the induction of cytokines by anti-CLEC9a hybridomas. LKcells expressing CLEC9a (10⁵ cells/well) were cultured with theindicated hybridomas before collecting the supernatants and test forIL-2.

FIG. 10 shows that plated anti-CLEC9a induces NFAT activity through theWT tail of CLEC9a in the presence of Syk and dependent on the presenceof Y7. B3Z expressing CLEC9a-CD3ζ (σ), or B3Z-Syk expressing CLEC9a-WT(ν) or CLEC9a-Y7(λ). NFAT activity is measured as indicated underMethods. The X axis shows the concentration of anti-CLEC9a antibodies(μg/ml) used to coat the plates.

FIG. 11 shows that anti-Clec9a antibodies immobilised on plastic induceproduction of IL12-23 p40 protein in Flt3L BMDC. Flt3L BMDC wereincubated on plates coated with anti-CLEC9a mAb and supernatants afterovernight culture were analyzed for IL12-23 p40.

FIG. 12 shows targeting of CD11c+CD8+ in vivo using anti-CLEC9a-S1 mAb.S1 conjugated anti-CLEC9a or isotype control mAb (5 μg) were injectedi.v. and the splenocytes processed the following day as indicated underMethods. A and B. CD11c positive and negative splenocytes were culturedfor four days with CFSE-labelled OT-I cells. A. CFSE profiles of OT-Icells in the presence of 25×10³ targeted CD11c− or CD11c+ splenocytes.B. Left panel: absolute number of OT-I cells per well. Right panel:IFN-γ production in the supernatants of expanded OT-I cells.

C and D. CD11c+ splenocytes in S1-anti-CLEC9a targeted mice were sortedas CD11c+B220-CD8+(CD8+ DC), CD11c+B220-CD4+ (CD4+ DC) andCD11c+B220−CD4−CD8− (DN DC) and cultured three days with CFSE labelledOT-I cells. C. CFSE profiles of OT-I cells in the presence of 25×10³targeted CD8+, DN, CD4+ DC. D. Left panel: absolute number of OT-I cellsper well. Right panel: IFN-γ production in supernatants at the end ofthe culture.

FIG. 13 shows induction of CTL immunity by targeting using anti-CLEC9amAb. A: S1 conjugated anti-CLEC9a or isotype control mAb were injecteds.c. together or not with anti-CD40 as indicated under Methods. Fivedays later target cells (congenic CD45.1) loaded with 20 nM (0.03 μMCFSE), 200 nM (0.3 μM CFSE) of SIINFEKL or not loaded with peptide (3 μmCFSE) were i.v. injected. Results are expressed as arithmetic mean±SEMof % specific lysis of the high dose of peptide in the in vivo killingassay (n=5, p<0.001 of DEC205 and CLEC9a groups compared to control. Oneway ANOVA). B: Results of a similar experiment using S2 conjugated toisotype control or anti-CLEC9a, administered with or without anti-CD40.

FIG. 14 shows the therapeutic effect of anti-CLEC9a+ anti-CD40 in thetreatment of B16 melanoma. A. time course of a tumor therapy experimentusing B16-OVA-GFP melanoma cells. Tumor cells (2×10⁵) were injected atday 0, treatment of Ab-S1+anti-CD40 performed at day 6, and lungsextracted and tumors counted at day 18. B. Tumor counts in each mouse inone representative experiment are shown. The reduction in tumor burdenis significant (p<0.001, one way ANOVA) with anti-CLEC9a or anti-DEC205plus anti-CD40.

FIG. 15 shows that human CLEC9a expression is restricted to BDCA-3+blood DC. (A) Human PBMC were stained with anti-hCLEC9a (8F9) or anisotype-matched control antibody (mouse IgG2a) and counterstained forvarious blood leukocyte markers. Histograms show CLEC9a staining on Tcells, B cells, monocytes, NK cells, lineage-negative HLA-DR− cells andlineage-negative HLA-DR+ cells. Number indicates the percentage ofhCLEC9a+ cells in the latter fraction. (B) PBMC from (A) were gated onlineage-negative HLA-DR+ cells. Dot plots show staining with anti-CLEC9aor isotype-matched control mAb against various blood DC subset markers.Numbers represent % cells in each quadrant. Specific staining is seenonly on BDCA-3+ DC. One representative experiment out of four.

FIG. 16 shows immunotherapy of B16 melanoma via targeting of tumorantigens to CLEC-9a. (A) Tumor therapy experiments were carried out asdepicted (upper left) using peptides encompassing known epitopes ofmelanocyte differentiation endogenous antigens (“Endo”: gp100, TRP-1 andTRP-2) covalently coupled to anti-CLEC9a or to an isotype-matchedcontrol antibody. Poly I:C+ anti-CD40 was used as adjuvant. Left lowerpanel shows representative pictures of lungs from mice treated asindicated. Right panel shows quantification of lung tumors in eachmouse. Data are pooled from two independent experiments (n=9 mice/group)and each point represents one mouse. (B) Splenocytes from individualmice in (A) were restimulated in vitro with the melanocytedifferentiation antigen peptides used for immunization (10 μM). IFN-γlevels after 2 d of culture are shown. Data pooled from two independentexperiments (n=9 mice/group). p values were calculated using the WhitneyU test. (C) Experiments were carried out as in FIG. 16 except that thevaccine was given one day prior to infusion of B16 cells. Data show thenumber of lung tumors per mouse. Data are pooled from two independentexperiments (n=7 mice/group) and each point represents one mouse. pvalues were calculated using the Mann Whitney U test.

FIG. 17 shows that CLEC9A binds a ligand on dying/dead cells. (A) Basalactivation of BWZ-mouse and human CLEC9Aζ reporter correlates withnumber of dead TO-PRO3⁺ cells. BWZ cells expressing a reporter for NFATcoupled to LacZ and stably expressing a chimeric molecule with theextracellular domain from mouse or human CLEC9A or control Dectin-1 andthe intracellular tail from CD3 were generated for screening for naturalligands of CLEC9A. Different cell concentrations were allowed to growfor two days, showing different degrees of overgrowth and increase ofdead cells in the culture tracked by TO-PRO 3 staining. The same amountof living BWZ cells was then plated in fresh medium and, after overnightculture, NFAT activity in BWZ cells was measured in a colorimetric assayas indicated in Methods.

(B) UVC-treated dead cells expose a ligand for CLEC9A. Live MEFs (Ctrl)or MEFs treated with UVC and left 24 h to induce cell death werecultured with BWZ NFAT reporter cells expressing the chimeric mouseCLEC9A-ζ, human CLEC9A-ζ, and Dectin-1-ζ. Where indicated, monovalentFab fragments of control (p21) or anti-mCLEC9A (1F6) or anti-hCLEC9A(8F9) were added to the culture. NFAT activity in BWZ cells was measuredas in (A).

(C) Dose response in UV-treated cells. LK cells were exposed todifferent doses of UVC as indicated in Methods and left 24 h to inducecell death before culturing with BWZ cells expressing mCLEC9A-ζ chimeraor control BWZ. NFAT activity in BWZ cells was measured as in (A).

(D) The recombinant soluble C-Type Lectin Domain (rsCTLD) of CLEC9Aselectively recognizes a molecule exposed by TO-PRO3⁺ dead cells.PE-tetramers of the rsCTLD of CLEC9A or Dectin-1 (as a control) wereused for staining of UV irradiated immortalized MEF (dot plots).Histograms show staining of zymosan, positive for Dectin-1 rsCTLD.

(E) Different death-inducing treatments trigger BWZ-CLEC9Aζ reportercells. BWZ-CLEC9Aζ cells were cultured overnight alone (Ctrl) or with LKcells untreated (LK) or treated with UVC (UV), mitoxantrone (Mtx), serumdeprivation (SD), osmotic shock (OS) or freeze and thaw (FT). BWZreporter activity (left y axis) and to-pro 3⁺ and CLEC9A rsCTLD⁺frequency in LK cells when starting the co-culture (right y axis) aredepicted.

(F) Staining of dead MEFs using PE-tetramers of CLEC9A rsCTLD. MEFs weretreated with UV and left 24 h or treated with osmotic shock (OS) beforestaining.

(A)-(F) one representative experiment shown of at least three performed.

FIG. 18 shows that CLEC9A is involved in cross-priming to dying cells byCD8α⁺ DC in vitro.

(A), (B) Blockade of CLEC9A results in impaired cross-priming of OT-Icells to dead cell associated antigen. (A) OT-I OVA-specific T cellswere cultured with CD8α-like Flt3L BMDC stimulated with OVA-loaded deadbm1 splenocytes in the presence or absence of anti-CLEC9A Fab (1F6) orcontrol Fab (p21). Three days later, proliferation (absolute numbers ofOT-I cells), IL-2 and IFNγ production (% production relative tountreated control) were measured. One representative experiment out ofsix is shown. (B) The average IL-2 and IFNγ production for the sixindependent experiments performed is shown as % production by untreatedcontrol for anti-CLEC9A Fab (1F6) or control Fab (p21).

(C) CLEC9A^(−/−) and WT CD8α-like Flt3L BMDC have the same ability tocapture dying cell material. WT or CLEC9A^(−/−) CD8α-like Flt3L BMDCwere incubated for 2 h with PKH26-labelled and UVC-treated bm1splenocytes at different ratios. Binding (4° C.) and binding+uptake (37°C.) were then quantified by flow cytometry for each type of DC.

(D) Impaired expansion and differentiation to effector OT-I cells afterincubation with CLEC9A^(−/−) Flt3L BMDC stimulated with OVA-expressingor OVA-loaded bm1 dead cells. CLEC9A^(−/−) Flt3L BMDC were cultured withUVC-treated OVA-loaded bm1 splenocytes or UVC-treated OVA-expressing bm1MEFs. OVA-specific OT-I T cells were then added and, after 3 days ofco-culture, absolute numbers of OT-I cells (left panel) or IFN-γ (rightpanel) were measured. Results show the average±SEM of two mice pergroup. One representative experiment out of three performed is shown.

(E) Impaired differentiation of OT-I cells after incubation withCLEC9A^(−/−) Flt3L BMDC stimulated with OVA-expressing UV-dead bm1 MEFsas in (D). One representative mouse per group out of four analyzed usingthe same assay is shown.

FIG. 19 shows that CLEC9A senses immunogenic cell death to promotecrosspriming in vivo.

(A), (B) Blockade of CLEC9A reduces crosspriming to dead cell associatedantigen in vivo. Mice untreated or treated with anti-CLEC9A (1F6, 400μg/mouse) or isotype control (rat IgG1) were immunised with i.v. with0.75×10⁶ UV-irradiated bm1 MEFs expressing a truncated OVA-GFP fusionprotein. Six days later H2K^(b)-OVA peptide tetramer positive cells (A)and IFNγ production in response to SIINFEKL ex vivo (B) were measured asreadout for induction of CD8⁺ T cell effector response arising from theendogenous repertoire. Individual mice and average for onerepresentative experiment (out of three) is shown.

(C)-(E) CLEC9A deficiency reduces crosspriming to dead cell associatedantigen in vivo. CLEC9a−/− mice or control littermates were immunised asin (A). The frequency of OVA-specific endogenous CD8+ T cells (C-D) wasquantified as in (A). (C) each dot represents an individual mouse pooledfrom six independent experiments and normalized as indicated underMethods. (D) the average of tetramer-positive cells is represented ineach litter for CLEC9a^(−/−) and CLEC9a⁺ mice. (E) IFNγ production inresponse to SIINFEKL ex vivo was measured as in (B). Individual mice andaverage for one representative experiment (out of three) is shown.

FIG. 20 shows that ligand for CLEC9a is exposed followingfixation/permeabilization.

LK cells were fixed with 2% formaldehyde or 2% para-formaldehyde, andpermeabilized or not with Tween (0.5%) and Tx-100 (0.5%). Cells wereextensively washed and percentage of permeable cells quantified usingTo-pro 3. Fixed or fixed-permeabilized cells were co-cultured with BWZNFAT reporter cells expressing the chimeric mouse CLAC9a-CD3ζ or controland NFAT activity measured.

FIG. 21 shows that dead cells signal through CLEC9a wt cytoplasmic tail.

LK cells were exposed to different doses of UVC as indicated in Methodsand left 24 h to induce cell death before culturing with B3Z cellsstably transfected with CLEC9a wt or CLEC9a with the Tyr7 mutated to Phe(Y7F) and co-expressing or not Syk. NFAT activity in B3Z cells wasmeasured as described under Methods.

FIG. 22. Both Fab monovalent and full bivalent anti-CLEC9a antibodiesblock dead cell signals through CLEC9a.

LK cells treated with UVC and left 24 h to induce cell death werecultured with B3Z cells stably transfected with CLEC9a wt or CLEC9a withthe Tyr7 mutated to Phe (Y7F) and co-expressing Syk or B3Z-Dectin-1-ζ ascontrol. Where indicated, monovalent Fab fragments of control (p21) oranti-CLEC9a (1F6) or full bivalent antibodies, including isotypecontrols (rat IgG1, rat IgG2a) and anti-mCLEC9a (1F6, 397, 7H11) wereadded to the culture. NFAT activity in B3Z cells was measured asdescribed under Methods.

FIG. 23 shows proliferation of OVA-specific OT-II CD4 T cells inresponse to anti-CLEC9a-OVA323-339. Naive CFSE labelled OT-II CD4 Tcells were transferred i.v. into C57BL/6 mice. One day later OVA323-339peptide was inoculated s.c. in the paw either conjugated to anti-CLEC9aor to isotype control antibody. Spleen and draining lymph nodes werecollected three to four days later and in vivo proliferation of OT-IIcells was tracked following CFSE dilution. Absolute numbers of OT-IIcells are shown, normalized between samples.

FIG. 24 shows that addition of adjuvant during the targeting in vivoinduces a strong Th1 response. Targeting in vivo in the presence orabsence of adjuvant (OT-II system). Mice were treated as in FIG. 23.Upper panel: Absolute number of OVA-specific CD4 T cells (afternormalization). Lower panels: IFN-γ production in the OT-II CD4 T cellspopulation expressed as percentage (left) or absolute number (right,after normalization). The effects on proliferation and differentiationobserved in the presence of poly I:C are not restricted to thisadjuvant. Curdlan has a similar effect.

FIG. 25 shows that the CD4 T cell response can be modulated by the typeof adjuvant/immunomodulator co-injected with the targeting reagent.Targeting in vivo in the presence or absence of adjuvant. OT-II systemwas performed as in FIG. 24. Upper panels: IL-17 production in the OT-IICD4 T cells population expressed as percentage (left) or absolute number(right, after normalization). Curdlan, a Dectin-1 agonist, acted as astrong adjuvant for Th17 polarization when administered with thetargeted antigen.

FIG. 26 shows that targeting in the absence of adjuvant leads toantigen-specific tolerance. Naive OVA-specific DO.11.10 CD4 T cells weretransferred into BALB/c mice. One day later, anti-CLEC9a-OVA 323-339 wasinoculated s.c. in the paw with or without adjuvant (poly I:C). Atdifferent time-points after immunisation, the size of the splenicDO.11.10 compartment was determined by flow cytometry using the KJ.126clonotypic antibody. Results are expressed as absolute number ofOVA-specific KJ.126 cells (after normalization) or as percentage ofKJ.126⁺ cells among the CD4 T cell compartment. At day 20, half of themice were challenged s.c. with OVA in Complete Freund's Adjuvant. 5 dayslater, the DO.11.10 response was monitored by flow cytometry. In miceinjected only with PBS at day 0, the remaining DO.11.10 cells are stillresponsive as shown by their strong proliferative responses afterrechallenge. A stronger response was detected in mice previouslyinjected with anti-CLEC9a+ adjuvant, showing that this firstimmunization probably led to the generation of memory cells. Incontrast, no response was detected in mice firstly immunized withanti-CLEC9a alone, showing that the remaining cells were tolerant forthe antigen

DETAILED DESCRIPTION OF THE INVENTION

CLEC9a

CLEC9a is a C-type lectin expressed on dendritic cells. As used in thisspecification, the term CLEC9a is intended to embrace the human protein(nucleic acid and protein sequences as shown in FIG. 1), the murineprotein (nucleic acid and protein sequences shown in FIG. 2), theirhomologues (especially orthologues) in other species, and variants andderivatives thereof which retain CLEC9a activity. Such variants andderivatives preferably have at least about 30% sequence identity, morepreferably at least about 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90% or 95% sequence identity to the human protein sequenceshown in FIG. 1, or at least about 35% identity, more preferably atleast about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95%identity to the extracellular domain of the human protein sequence shownin FIG. 1.

In particular, conservative substitutions in the CLEC9a sequence (ascompared to the reference sequences) may be particularly well tolerated,without substantial effect on function.

A conservative substitution may be defined as a substitution within anamino acid class and/or a substitution that scores positive in theBLOSUM62 matrix.

According to one classification, the amino acid classes are acidic,basic, uncharged polar and nonpolar, wherein acidic amino acids are Aspand Glu; basic amino acids are Arg, Lys and His; uncharged polar aminoacids are Asn, Gln, Ser, Thr and Tyr; and non-polar amino acids are Ala,Gly, Val, Leu, Ile, Pro, Phe, Met, Trp and Cys.

According to another classification, the amino acid classes are smallhydrophilic, acid/acid amide/hydrophilic, basic, small hydrophobic andaromatic, wherein small hydrophilic amino acids are Ser, Thr, Pro, Alaand Gly;

acid/acidamide/hydrophilic amino acids are Asn, Asp, Glu and Gln; basicamino acids are His, Arg and Lys; small hydrophobic amino acids are Met,Ile, Leu and Val; and aromatic amino acids are Phe, Tyr and Trp

Substitutions which score positive in the BLOSUM62 matrix are asfollows:

Original C S T P A G N D E Q H R K M I L V F Y W Residue Substitution —T S — S — S N D E N Q E I M M M Y H F A D E Q R Y K Q L L I I W F Y N HK K R V V V L W

Percent (%) amino acid sequence identity with respect to a referencesequence is defined as the percentage of amino acid residues in acandidate sequence that are identical with the amino acid residues inthe reference sequence, after aligning the sequences and introducinggaps, if necessary, to achieve the maximum percent sequence identity,and not considering any conservative substitutions as part of thesequence identity. % identity values may be determined by WU-BLAST-2(Altschul et al., Methods in Enzymology, 266:460-480 (1996)). WU-BLAST-2uses several search parameters, most of which are set to the defaultvalues. The adjustable parameters are set with the following values:overlap span=1, overlap fraction=0.125, word threshold (T)=11. A % aminoacid sequence identity value is determined by the number of matchingidentical residues as determined by WU-BLAST-2, divided by the totalnumber of residues of the reference sequence (gaps introduced byWU-BLAST-2 into the reference sequence to maximize the alignment scorebeing ignored), multiplied by 100.

A CLEC9a agonist is an agent capable of inducing CLEC9a activity,typically by binding to its extracellular domain (ECD) and inducingintracellular signalling via its intracellular domain. Signalling mayinvolve one or more of binding of Syk to the intracellular domain,phosphorylation (and hence activation) of Syk, and/or phosphorylation ofErk and/or activation of NFAT. An illustrative assay is described in theExamples, using B3Z cells transfected with Clec9a and Syk. The skilledperson will understand that a chimeric protein having the extracellulardomain of Clec9a and an intracellular domain derived from a differentprotein may also be used to assay for Clec9a agonist activity. Thetransmembrane domain may be from Clec9a, the same protein as theintracellular domain, or from another protein. An example is theCD3ζ-NKRP1-CLEC9a illustrated in FIG. 5 and described in more detail inthe Examples.

A CLEC9a antagonist is an agent capable of inhibiting or blocking CLEC9afunction. For example, it may prevent its normal expression, its abilityto bind physiological CLEC9a ligand, its ability to internalise(endocytose) molecules to which it has bound, its ability to signalintracellularly (see above), or the ability of its ECD to interact withbinding partners (ligands or receptors), e.g. on other cells. Anotherpossible mechanism of action for an antagonist might be to promoteinternalisation of Clec9a without inducing significant intracellularsignalling, and so reduce the pool of Clec9a available at the cellsurface to interact with natural ligands or other agonists. Antagonistsinclude binding agents having affinity for CLEC9a such as anti-Clec9aantibodies which lack significant agonist activity. These may bereferred to as “blocking” antibodies. Monovalent or bivalent antibodieswithout agonist activity may be particularly suitable as blockingantibodies. Other Clec9a antagonists include nucleic acid molecules oranalogues thereof capable of hybridising with DNA or RNA encodingCLEC9a. Such agents include ribozymes, RNAi, siRNA, etc.

Further CLEC9a antagonists are competitors for the CLEC9a ligand, whichcan block binding sites on the ligand for dendritic cell-associatedCLEC9a and so prevent the ligand from being recognised or bound by thedendritic cell. Suitable competitors include soluble moleculescomprising the extracellular domain of CLEC9a or a portion thereofsufficient to bind to the CLEC9a ligand. Thus the molecule may comprisean amino acid sequence having at least 70% identity, at least 75%identity, at least 80% identity, at least 85% identity, at least 90%identity, or at least 95% identity to the extracellular domain (CTLD) ofhuman CLEC9a as shown in FIG. 1, or murine CLEC9a as shown in FIG. 2, ora fragment thereof having affinity for the CLEC9a ligand. The fragmentmay comprise at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 110 or atleast 120 amino acids of the respective extracellular domain sequence ora sequence having the required level of identity therewith.

The CLEC9a extracellular domain (or portion thereof) may be associatedwith a heterologous moiety which may modulate some property of theantagonist, such as its pharmacokinetic properties in vivo. Theextracellular domain may be covalently or non-covalently bound to theheterologous moiety, or may be expressed as a fusion protein with theheterologous moiety. For example, the heterologous moiety may be anantibody Fc domain, in order to provide increased serum half life andallow efficient clearance of complexes between the antagonist and theCLEC9a ligand.

Other possible functions of the heterologous moiety include mediatingoligomerisation of the CLEC9a extracellular domain, and facilitationpurification of the antagonist or isolation from a sample. For example,a suitable antagonist may be a soluble molecule comprising or consistingof the CLEC9a extracellular domain (or a fragment thereof sufficient tobind CLEC9a ligand) associated with an avidin monomer. The avidinmonomers will tend to associate into tetramers, providing a complexcomprising four CLEC9a domains and four avidin subunits. This constructcan readily be isolated by contact with biotin, which may be provided ona solid support such as a bead.

Where the binding agent or antagonist is a protein, it may be possibleto administer a nucleic acid (e.g. DNA) encoding the antagonist.Typically the nucleic acid will be taken up by cells within the body(e.g. muscle cells), expressed, and secreted from those cells. Thisapproach is often referred to as DNA vaccination.

Antibodies are particularly suitable as binding agents and antagonists,and can conveniently be expressed in scFv form. If necessary, anantibody can be encoded as a fusion protein with the antigen, or with aneffector moiety as described above. An example of a DNA vaccinationapproach is described in Nchinda et al., J. Clin. Invest. 118(4),1427-36, 2008.

The nucleic acid typically comprises a coding region encoding thebinding agent or antagonist, optionally in conjunction with any desiredfusion partner, in operable linkage with transcriptional andtranslational regulatory sequences to ensure appropriate expression andsecretion of the protein from cells which take up the nucleic acid. Suchsequences include (but need not be limited to) transcriptionalinitiation sequences (e.g. promoter and enhancer), transcriptionaltermination sequences, appropriate splicing signals, translationalinitiation and termination sequences, and a signal peptide to enablesecretion.

Thus the invention further provides a nucleic acid (e.g. a DNA) encodinga CLEC9a antagonist or binding agent, for use in a method of medicaltreatment. Also provided is a nucleic acid encoding a CLEC9a antagonistor binding agent for use in a method of and therapeutic uses thereof.

CLEC9a Ligand

The present inventors have found that CLEC9a recognises a liganddisplayed by certain types of dead and dying mammalian cells. Inparticular, certain types of cell death appear to trigger display of theligand. This is a surprising finding because many known members of theC-type lectin family (including Dectin-1, which is the most closelyrelated protein to CLEC9a) are receptors for pathogen-associatedmolecular patterns, and so recognise structures displayed by pathogens,rather than self molecules.

It is well recognised that certain mechanisms of self cell death arecapable of triggering an immune response. These may be regarded asimmunogenic cell death. It has been proposed that death by apoptosis(which normally does not result in rupture of the plasma membrane andrelease of the intracellular contents) is non-immunogenic, while deathby other mechanisms such as necrosis (which do involve rupture of theplasma membrane and release of the cell contents) is immunogenic.However, the physiological situation appears to be rather more complexthan this. For example, apoptotic cells in vivo are normally absorbed(phagocytosed) by neighbouring cells such as macrophages before theprocess of cell death is complete. However, if the cells are notphagocytosed, so-called secondary necrosis may occur, in which theplasma membrane may be disrupted and cellular contents released. Celldeath of this nature may be immunogenic, despite being apoptotic atleast in part.

Immunogenic cell death may play a role in the onset, development orpersistence of autoimmune disease. This is reviewed, for example, byVioritto et al (Clin Immunol 122(2), 125-134 (2007)), Tesniere et al(Curr Op Immunol 21, 1-8 (2008)) and Kim et al (Immunity 27, 321-333(2007))

Dendritic cells may play a role in induction of any immune responsecaused by immunogenic cell death, by taking up cellular debris from thedead or dying cells (or even absorbing the entire cell) and presentingprocessed fragments to T cells.

The present inventors have now found that CLEC9a is capable of bindingto a ligand displayed by dead or dying cells, and that CLEC9a signallingmay be triggered by this interaction.

It is also believed that the ligand is not synthesised de novo duringthe process of cell death. Rather, it may be constitutively expressed bysome or all mammalian cells but is not accessible for interaction withCLEC9a while the cell remains healthy. Certain types of cell deathresult in exposure of the ligand and/or release of the ligand from thecell, in a form capable of interacting with CLEC9a. This may involvedisruption of the plasma membrane.

Experimentally, exposure of the ligand can be caused by treatments suchas irradiation (e.g. with ionising radiation such as UV light), serumdeprivation, at least one freeze/thaw cycle, or by treatment withchemotherapeutic agents such as anthracyclines (such as doxorubicin anddaunorubicin) and anthracenedione (such as mitoxantrone). However deathby osmotic shock appears not to expose the ligand.

Thus, without wishing to be bound by any particular theory, it isbelieved that CLEC9a may be involved in generation of the immuneresponse caused by immunogenic cell death. The interaction betweenCLEC9a and its ligand can be inhibited by CLEC9a antagonists. Theseinclude binding agents capable of binding to (the extracellular domainof) CLEC9a. Other examples include competitors for CLEC9a binding siteson the ligand, such as soluble agents comprising the extracellulardomain of CLEC9a or a portion thereof (e.g. at least 20 amino acids, atleast 50 amino acids, at least 100 amino acids, at least 150 aminoacids, or at least 200 amino acids of the extracellular domain) which iscapable of binding to CLEC9a ligand.

Antagonists capable of inhibiting binding between CLEC9a and its ligandwill be capable of inhibiting CLEC9a signalling when contacted withsuitable dead or dying cells (e.g. UV-irradiated mammalian cells) or alysate, extract or fraction thereof capable of inducing CLEC9asignalling. Any suitable test system may be used to assess thiscapacity. For example, in dendritic cells expressing CLEC9a, CLEC9asignalling may be assessed by determining phosphorylation of Syk kinase.Alternatively an artificial reporter system may be used comprising theCLEC9a extracellular domain functionally linked to a reporter systemsuch as the CD3zeta chimera described in the examples.

Binding Agents

Any suitable molecule having a sufficiently high affinity andspecificity for CLEC9a may be used as a binding agent. The molecule maybe a protein, nucleic acid (e.g. an aptamer), carbohydrate (e.g. oligo-or polysaccharide), small molecule, etc. Particularly preferred bindingagents are physiological ligands for CLEC9, and antibodies againstCLEC9a and functional fragments thereof.

The binding agent preferably has a binding affinity (affinity constant)for CLEC9a, particularly for the CLEC9a ECD, of at least 10⁵M⁻¹, atleast 10⁶M⁻¹, at least 10⁷M⁻¹, preferably at least 10⁸M⁻¹, morepreferably at least 10⁹M⁻¹.

The binding agent preferably has an affinity at least 2×, and preferablyat least 5×, at least 10×, at least 50× or at least 100× greater thanfor any non-CLEC9a molecule, including other C-type lectins.

It is well-known that fragments of a whole antibody can perform thefunction of binding antigens. Examples of functional binding fragmentsare (i) the Fab fragment consisting of VL, VH, CL and CH1 domains; (ii)the Fd fragment consisting of the VH and CH1 domains; (iii) the Fvfragment consisting of the VL and VH domains of a single antibody; (iv)the dAb fragment (Ward, E. S. et al., Nature 341, 544-546 (1989)) whichconsists of a VH domain; (v) isolated CDR regions; (vi) F(ab′)2fragments, a bivalent fragment comprising two linked Fab fragments (vii)single chain Fv molecules (scFv), wherein a VH domain and a VL domainare linked by a peptide linker which allows the two domains to associateto form an antigen binding site (Bird et al, Science, 242, 423-426,1988; Huston et al, PNAS USA, 85, 5879-5883, 1988); (viii) bispecificsingle chain Fv dimers (PCT/US92/09965) and (ix) “diabodies”,multivalent or multispecific fragments constructed by gene fusion(WO94/13804; P. Holliger et al Proc. Natl. Acad. Sci. USA 90 6444-6448,1993).

As antibodies can be modified in a number of ways, the term “antibody”should therefore be construed as covering any specific binding substancehaving an binding domain with the required specificity. Thus, this termcovers the antibody fragments described above, as well as derivatives,functional equivalents and homologues of antibodies, including anypolypeptide comprising an immunoglobulin binding domain, whether naturalor synthetic. Chimaeric molecules comprising an immunoglobulin bindingdomain, or equivalent, fused to another polypeptide are thereforeincluded. Cloning and expression of chimaeric antibodies are describedin EP-A-0120694 and EP-A-0125023.

It will be appreciated that the binding agents used in the methodsdescribed herein are generally required to bind the extracellular domainof CLEC9a in order to exert the required effect. Reference to a bindingagent capable of binding CLEC9a should be construed accordingly, unlessthe context allows otherwise.

In certain aspects of the invention, it is desirable to cross-link anantigen (e.g. a protein or peptide antigen) to a binding agent asdescribed. The skilled person is well aware of suitable methods andreagents. Where the binding agent is a protein, the antigen may becoupled via a sulphydryl group of the binding agent. The sulphydrylgroup may normally be free, or it may normally be part of a disulphidebond in which case it may be exposed by selective reduction of thebinding agent. For example, an antibody can be mildly reducedselectively in the hinge region using the reducing agentmercaptoethanosulfonate. Then, the antigen is activated usingsulpho-SMCC, an hetero-bifunctional cross-linking reagent that reactswith the tertiary amines of the protein, generating groups reactive withfree sulphydryls. Then, the antibody and the activated antigen areincubated together resulting in the protein being conjugated to themonovalent antibody¹⁸. Alternatively, if a suitably immunogenic peptidesequence from the antigen is known, such a peptide containing a cysteinewith a free sulphydryl can be synthesized and coupled to sulpho-SMCCactivated antibody, which will remain bivalent and with several peptidesbound per molecule of antibody.

Pharmaceutical Compositions

The polypeptides, antibodies, peptides, nucleic acids and cellsdescribed herein can be formulated in pharmaceutical compositions. Thesecompositions may comprise, in addition to one of the above substances, apharmaceutically acceptable excipient, carrier, buffer, stabiliser orother materials well known to those skilled in the art. Such materialsshould be non-toxic and should not interfere with the efficacy of theactive ingredient. The precise nature of the carrier or other materialmay depend on the route of administration, e.g. oral, intravenous,cutaneous or subcutaneous, nasal, intramuscular, intraperitoneal routes.

Pharmaceutical compositions for oral administration may be in tablet,capsule, powder or liquid form. A tablet may include a solid carriersuch as gelatin or an adjuvant. Liquid pharmaceutical compositionsgenerally include a liquid carrier such as water, petroleum, animal orvegetable oils, mineral oil or synthetic oil. Physiological salinesolution, dextrose or other saccharide solution or glycols such asethylene glycol, propylene glycol or polyethylene glycol may beincluded.

For intravenous, cutaneous or subcutaneous injection, or injection atthe site of affliction, the active ingredient will be in the form of aparenterally acceptable aqueous solution which is pyrogen-free and hassuitable pH, isotonicity and stability. Those of relevant skill in theart are well able to prepare suitable solutions using, for example,isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection,Lactated Ringer's Injection. Preservatives, stabilisers, buffers,antioxidants and/or other additives may be included, as required.

Administration is preferably in a “prophylactically effective amount” ora “therapeutically effective amount” (as the case may be, althoughprophylaxis may be considered therapy), this being sufficient to showbenefit to the individual. The actual amount administered, and rate andtime-course of administration, will depend on the nature and severity ofwhat is being treated. Prescription of treatment, e.g. decisions ondosage etc, is within the responsibility of general practitioners andother medical doctors, and typically takes account of the disorder to betreated, the condition of the individual patient, the site of delivery,the method of administration and other factors known to practitioners.Examples of the techniques and protocols mentioned above can be found inRemington's Pharmaceutical Sciences, 20th Edition, 2000, pub.Lippincott, Williams & Wilkins.

A composition may be administered alone or in combination with othertreatments, either simultaneously or sequentially dependent upon thecondition to be treated.

EXAMPLES

CLEC9a Sequences and Structure

A search of the NCBI gene database shows that CLEC9a sequence has beenalready identified in Mus musculus, Pan troglodytes, Homo sapiens andMacaca mulatta. A blast search using the protein sequence of mouseCLEC9a, also shows predicted CLEC9a proteins in Rattus norvegicus, Canisfamiliaris and Bos Laurus. The human cDNA sequence and the annotatedprotein sequence with relevant domains is detailed in FIG. 1. The mousecDNA sequence which we have cloned from mouse CD8α+ DCs and itsannotated protein sequence is detailed in FIG. 2. This sequence differsfrom the published cDNA sequence, which contains an additional G residuecausing a frameshift towards the end of the molecule, leading to alonger protein than that shown in FIG. 2. Our sequence appears to becorrect, since it matches the published genomic sequence (NC_(—)000072.4GI:94471533); as can be seen in this page the position 13480 of thegenomic (ATTT) matches our cDNA sequence. The sequences predict a C-typelectin family protein with a C-type lectin-like domain, (CTLD), a stalkregion, a transmembrane region and a cytoplasmic domain containingone-species-conserved tyrosine highlighted in FIGS. 1 and 2. We havefound transcripts for three isoforms of mouse CLEC9a that we have termedlong isoform (exons 1-7), short isoform that lacks exon 4, including aputative cysteine involved in dimerization, and very short isoform, thatcouples exon 3 to exon 7, yielding a mRNA coding for a transmembraneprotein that, if expressed, would share the transmembrane-intracellulardomains with CLEC9a but would have a short and different extracellulardomain. We only have evidence of protein expression for the longisoform.

The structure of mouse CLEC9a was analyzed. The core protein has apredicted molecular weight (Mw) of about 29.67 KDa However, whenexpressed in the HEK-293 cell line the Mw is about 100 KDa innon-reducing conditions, and a Mw of about 45 KDa in reducing conditions(FIG. 3). These results indicate that the molecule forms dimers throughthe cysteine in the stalk, like other lectins in the family, and themonomer is strongly glycosylated.

CLEC9a Expression

CLEC9a was first detected in our laboratory as a result of arepresentational difference analysis between samples of mouse spleenCD11c+CD8+ and CD11c+CD8− cells. The results showed that sequencescorresponding to the EST clone AW318446, corresponding to CLEC9a, wereselectively found in the CD11c+CD8+ transcripts.

The analysis of the transcripts in sorted subsets of splenic DCsrevealed high expression of CLEC9a in the CD8+ subset, although doublenegative (CD4−CD8−) and B220+ spleen DC also showed some transcripts forCLEC9a (FIG. 4). No significant expression was found in GMCSF-derivedBMDC (FIG. 4). Mouse bone marrow cultured for 10 days in the presence ofFlt3L (50 ng/ml) generates CD11c+ cells that are either CD11b+,functionally corresponding to spleen conventional DC and including aCD8-like subset¹⁹, or B220+, which are functional equivalents ofplasmacytoid DC (pDC). High expression of CLEC9a was found in the sortedCD11b+ subset, although the pDC subset showed some expression of themolecule (FIG. 4).

Since the RT-PCR is a sensitive technique that can detect very low levelof transcript, it is limited by the quality of purification of thesample. To unequivocally determine the pattern of expression of CLEC9a,we generated rat monoclonal antibodies (mAb) against mouse CLEC9a, usinga CD3ζ-NKRP1-CLEC9a chimera expressed in B3Z cells with a β-Gal reporter(FIG. 5) as indicated under Methods. We selected three mAbs named 1F6,397, and 7H11. Using these mAb, we have studied the pattern ofexpression of the molecule in mouse spleen and bone marrow. CLEC9a washighly expressed in CD8α+ conventional DCs (MFI˜350-400) and showedmoderate expression (MFI˜65-70) in pDCs (FIG. 6). The molecule was notdetected in other cell types explored including B cells, T cells, NKcells, NKT cells, monocytes, macrophages and granulocytes.

As a model for analysis of CLEC9a function in vitro, we analyzedexpression in mouse GMCSF- and Flt3L-derived BMDC. We did not detectexpression of the molecule in GMCSF-derived BMDC, whereas CLEC9a wasselectively expressed in the CD11b^(lo), CD24^(hi), B220− subset ofFlt3L BMDC, functionally homologous to CD8+ splenic DC¹⁹, and also in asubset of the CD11b^(lo) B220+, equivalent to pDC (Data not shown).

CLEC9a Signals Through Syk Kinase and Promotes DC Activation.

Sequence analysis reveals Tyr7 from mouse that is conserved in all thespecies with the same structure EXXYXXL, which could serve as a putativeSH2 binding domain and/or a tyrosine-based sorting signal. This sequenceallows/mediates Syk binding in mouse Dectin-1 and is termed “HemITAM”²².However, this putative sequence is necessary but not sufficient forpredicting Syk binding. In consequence, we designed biotinylatedpeptides with the cytoplasmic tail of mouse CLEC9a expressing thephosphorylated Tyr7, or with that Tyr7 without phosphorylation or evenmutated to Phe. The Tyr7 phosphorylated peptide was able to pull downrecombinant Syk (FIG. 7) to a similar extent than the cytoplasmic tailfrom mouse Dectin-1 expressing both Tyr phosphorylated as a positivecontrol²³.

To determine if CLEC9a truly acts as a signalling receptor, we used therat anti-mouse CLEC9a mAb above described. We generated transfectantsexpressing CLEC9a in LK cells, a mouse B cell line negative for CLEC9aexpression but containing endogenous levels of Syk²³. The triggering ofCLEC9a with all three plated anti-CLEC9a antibodies tested resulted inphosphorylation of Syk and Erk in LK cells (FIG. 8).

To determine whether Syk is necessary for CLEC9a signalling, we usedstable transfectants of the reporter T cell line B3Z, which does notexpress Syk. The contribution of the Tyr7 to CLEC9a signalling wasanalyzed using a mutant version of CLEC9a with the Tyr7 mutated to Phe(CLEC9a Y7F). We transduced B3Z cells with CLEC9a wt or Y7F andco-transduced or not with Syk kinase. Plated anti-CLEC9a mAbs inducedNFAT activation in B3Z-C9 wt-Syk through the wt cytoplasmic tail of C9,but not in the absence of either Syk or the Y7 (not shown).

Regulation of Immuno-Regulatory Cytokines/Co-Stimulatory MoleculesThrough CLEC9a.

To determine whether signalling through CLEC9a can contribute to theproduction of regulatory cytokines or expression of co-stimulatorymolecules in the cells where is expressed, we have analyzed LKtransfectants with wt CLEC9 or the Y7F mutant. Hybridoma cellsexpressing 397 anti-CLEC9a and, to a lesser extent, 7H11 triggeredspecific IL-2 production through the CLEC9a molecule that was abolishedin the Y7F mutant (FIG. 9). DEC-205 control hybridoma did not triggerany response and 1F6 anti-CLEC9a triggered a reduced response that opensthe possibility that these antibodies can behave differentially forcytokine production, which would be very attractive from the prospectiveof CLEC9a targeting (agonist antibody, 397, versus blocking antibody,1F6).

To determine the requirements for CLEC9a WT tail activation we analyzedstable transfectants of the reporter T cell line B3Z, which does notexpress Syk. The contribution of the Tyr7 to CLEC9a signalling wasanalyzed using a mutant version of CLEC9a with the Tyr7 mutated to Phe(CLEC9a Y7F). We transduced B3Z cells with CLEC9a wt or Y7F andco-transduced or not with Syk kinase. Plated anti-CLEC9a mAbs inducedNFAT activation in B3Z-C9 wt-Syk through the wt cytoplasmic tail of C9,but not in the absence of either Syk or when the Y7F mutant was used(FIG. 10).

Flt3L BMDC respond to plated anti-CLEC9a mAbs by production of IL12-23p40 protein (FIG. 11).

These results demonstrate that CLEC9a is a signalling molecule capableof activating DCs.

CLEC9a is an Endocytic Receptor that Selectively Targets CD11c+CD8+DC InVivo

The potential of anti-CLEC9a mAb to be internalised by endogenous CLEC9aexpressed in Flt3L BMDC was analyzed by FACS and revealed that CLEC9a isan endocytic molecule. Confocal analyses showed targeting of theantibody to intracellular compartments (data not shown). These resultssuggest that CLEC9a is an endocytic receptor that can be targeted byantibodies coupled to antigens (tumor/viral vaccination) to specificallydeliver the cargo to cell subsets selectively expressing the molecule.To determine whether CLEC9a mAb serve as a targeting tool in vivo, weinjected i.v. 7H11-Alexa-488 or isotype control. After 16 h, we analyzedtotal splenocytes and the antibody selectively targeted CD8α+DC(MFI-350-400), and, with lower affinity, PDC (MFI-65-70). Labelling ofsplenocytes with anti-rat Cy5 suggested that most of the rat anti-mouseCLEC9a mAb was endocytosed, since it did not co-stained with theanti-rat secondary reagent (not shown).

To explore whether the targeting through CLEC9a leads to the processingand presentation of antigen by a specific subset in vivo, we coupled abiotinylated derivative of an immuno-dominant peptide for the CTLresponse to OVA protein (SIINFEKLC-biot (SEQ ID NO: 6), named 51) toisotype control or anti-CLEC9a mAb as indicated under Methods. Thebiotinylation of the peptide allowed us to determine that there wasbetween 1-1.2 peptides per antibody in all cases. Mice were injectedi.v. either with 5 μg of the S1-coupled anti-CLEC9a or with theS1-isotype control. The following day, splenocytes were enriched inCD11c+ or CD11c-subsets and tested for their ability to induce OT-I Tcell proliferation and cytokine production (FIGS. 12 a and b). Only theanti-CLEC9a targeted CD11c+ cells resulted in proliferation and IFN-γproduction by OT-I cells, showing that CLEC9a targets specificallyCD11c+ DC resulting in presentation to antigen-specific T cells.

To further determine which subset of dendritic cells is targeted byanti-C9, mice injected with S1-coupled anti-C9 were sorted in the threemajor subsets of conventional DCs and tested with OT-I T cells as above.Only the CD8₊ subset of DCs mediated proliferation and IFN-γ productionby T cells, confirming that CLEC9 targets specifically the CD11c+ CD8+DC resulting in priming of antigen-specific T cells (FIGS. 12 c and d).

Targeting In Vivo Using Anti-Clec9a mAb Plus Anti-CD40 Results inPriming of CTLs and Tumor Rejection

We explored whether CLEC9a targeting in vivo could induce specific Tcell activation. Injection of 2 μg of anti-C9-S1, but not the isotypecontrol, results in the induction of specific CTL activity from theendogenous repertoire in vivo when co-administered with anti-CD40 (FIG.13). This behaviour is similar to that observed with anti-DEC205-S1,previously described²⁴ as evidenced by in vivo killing assays (FIG.13A).

Mice given S1 coupled to control mAb did not eliminate target cellsirrespective of anti-CD40 co-administration. In contrast, target cellswere completely eliminated from mice given S1 coupled to anti-CLEC9atogether with anti-CD40. No response was seen when the anti-CD40 mAb wasomitted. Consistent with target cell elimination, significant numbers oftetramer positive OVA/H-2 Kb-specific CD8+ T cells were found only inthe spleens and blood of mice that had received anti-CLEC9a-S1 togetherwith anti-CD40. Re-stimulation of the same cells with SIINFEKL (SEQ IDNO: 7) peptide in vitro resulted in secondary expansion, with IFN-γproduction and specific killing activity. Identical results wereobtained using anti-CLEC9a conjugated to a longer peptide of OVAcontaining the SIINFEKL epitope (SEQ ID NO: 7;“S2”;SIINFEKLTEWTSSNVMEERC (SEQ ID NO: 8); FIG. 13E).

Notably, free S1 peptide was unable to induce in vivo killing responsesor elicit a significant number of tetramer positive cells even whengiven at 100 times excess over the amount present in anti-CLEC9a-S1conjugates (not shown). We conclude that targeting of exogenous antigento CLEC-9a together with an appropriate adjuvant allows efficientcrosspriming of CD8+ T cells.

To determine whether CLEC9a priming of CTL activity can result in tumortherapy, we used the model of B16 melanoma lung metastasis. Weinoculated i.v. 2×10⁵B16-OVA-GFP melanoma cells and 6 days laterdifferent antibodies conjugated to S1 SIINFEKL (SEQ ID NO: 7) derivative(10 μg) together with anti-CD40 (25 μg) were injected s.c. in the paw(FIG. 14). After 18 days following injection of the tumor, the number oflung tumors was analyzed. The results revealed that anti-CLEC9a plusanti-CD40 is effective for tumor therapy (p<0.001, one way ANOVA) (FIG.14).

We extended the experiments to determine whether anti-CLEC9a targetingcan also be used to induce immune responses to endogenous melanocytedifferentiation proteins that can serve as B16 tumor-associated antigens(25-27). We synthesised biotinylated peptides encompassing H-2 Kb andH-2 Db-restricted antigenic epitopes from gp100, TRP-1 and TRP-2(25-27), coupled these covalently to anti-CLEC-9a and immunized micewith the antibody conjugates together with poly I:C and anti-CD40 asadjuvants. As shown in FIG. 16, a single dose of vaccine giventherapeutically three days post transfer of B16 melanoma cells inducednearly complete eradication of lung pseudo-metastases. This wasaccompanied by the induction of potent IFN-γ responses against themelanoma antigens (FIG. 16). In contrast, the same antigens inuntargeted form (conjugated to a control isotype-matched mAb) failed toinduce protection or IFN-γ responses (FIGS. 16A, B). Similar resultswere obtained in a prophylactic model in which the vaccine was givenprior to B16 challenge (FIG. 16C). We conclude that priming of specificCTL via CLEC-9a targeting can be used for prophylactic or therapeuticvaccination against mouse tumors.

Human Clec9a Expression is Restricted to a Small Subset of Blood DC

To extend these findings to humans, we cloned hClec9a and generatedmouse mAbs against it (see Materials and Methods). One of these mAbs wasselected to analyze the pattern of Clec9a expression among humanperipheral blood mononuclear cells. Human Clec9a expression was absentfrom lymphocytes, monocytes, NK cells and lineage-negative HLADR-cells(FIG. 15A). It was also not detected in monocyte-derived DC generated byculture in GM-CSF and IL-4 (data not shown). However, Clec9a expressionwas apparent in a discrete subpopulation of blood DC, defined aslineage-negative HLA-DR+ cells (FIG. 15A).

Five distinct subsets of blood DC have been reported, including apopulation of CD123+ pDC and different subsets of putatively myeloidCD123− DC distinguishable on the basis of expression of CD16, CD1b/c,BDCA-3, and CD34 (22). The Clec9a+ subpopulation of DC was negative forCD123, suggesting that human pDC do not express Clec9a, unlike pDC inthe mouse (FIG. 15B). Clec9a+ blood DC were also negative for CD34, CD16and CD1b/c. However, Clec9a+ DC were uniformly positive for BDCA-3 (FIG.15B). Human Clec9a therefore selectively marks a distinct population ofBDCA-3+ DC.

Finally, we assessed whether human CLEC-9a, like its mouseorthologue/can function as an endocytic receptor in DC. BDCA-3+ DC werestained at 4° C. with Alexa 488-labelled anti-CLEC-9a. After 1 h at 37°C. but not at 4° C., fluorescence was found in intracellularcompartments (not shown). Therefore, human CLEC-9a mediates endocytosisof bound antibody in BDCA3+ DC, thereby suggesting that it could be usedfor antigen targeting to these cells in humans.

Dying/Dead Cells Express a Ligand for CLEC9a.

BWZ cells containing the CLEC9A-CD3ζ chimera have a short generationtime and can easily overgrow, generating a significant proportion ofdead cells in the culture. We observed a basal activation in the BWZtransfectants expressing CLEC9A without any further stimulation,correlating with the number of dead cells in the BWZ culture (FIG. 17a).

To confirm these results we used a cell line that does not induce aresponse when added to the BWZ-CLEC9Aζ transfectants. LK cells do notinduce a response when exposed to the reporter cells. However, when LKcells were UV-irradiated to induce cell death, they turned into potentinducers of the reporter (FIG. 17 b), suggesting that altered cellsfollowing UV treatment express ligand/s for CLEC9A. Since bivalentantibodies trigger cross-linking of the molecule expressed in the B3Zcell line and reporter activation, blocking antibodies cannot be testedin this system to demonstrate the specificity of the interaction.However, monovalent Fab fragments of anti-CLEC9A antibodies did nottrigger the reporter activity and blocked induction by UV-treated cellsin a species-specific fashion (FIG. 17 b), demonstrating that theantibody acts blocking specifically the CLEC9A receptor to avoidinteraction with the ligand.

This result was confirmed in other independent UV-irradiated cell types(murine 3T3, LK cells, MEFs, EL-4), rat RBL cells, and human HEK-293.When cells are pre-incubated with caffeine (which prevents UV-inducedDNA damage and apoptosis) and then exposed to UV radiation, exposure ofthe ligand is not induced.

We exposed LK cells to different doses of UV and we found thatexpression of ligand in LK cells correlates with the number of dead LKcells (FIG. 17 c), showing that the ligand is selectively expressed inthis population.

To confirm this in an independent fashion, we generated recombinantsoluble extracellular domain (rsCTLD) for mouse CLEC9A, and mouseDectin-1 as a control, each coupled to a BirA sequence formonobiotinylation. Monobiotinylated rsCTLD was used to generatePE-tetramers. We stained cells treated with UV 24 h earlier and wedetected specific binding of CLEC9A rsCTLD tetramers to TO-PRO 3positive cells (FIG. 17 d, dot plots). As a control, Dectin-1 rsCTLDtetramers did not bind dead cells, yet they bound to their specificligand zymosan (FIG. 17 d, histogram).

As UV treatment induces DNA damage and a series of related stressmarkers, we tested whether the induction of ligands was caused by DNAdamage or mostly by processes involved in cell death. Not only DNAdamage-causing reagents, but also serum deprivation or even freeze-thaw,which has been shown to promote primary necrosis resulting inimmunogenic cell death, led to exposure of the ligand (FIG. 17 e).However, osmotic shock, which induces instant cell death that has beenshown to behave as tolerogenic, did not expose CLEC9A ligand (FIGS. 17 eand f). In conclusion, CLEC9A ligand is exposed in cells followingcertain types of primary and secondary necrosis. Moreover, we have foundthat fixation (or fixation and permeabilization) of the cells instantlypromotes changes that make cells permeable to TO-PRO3 and “expose” theligands for CLEC9A (FIG. 20) further demonstrating that synthesis of theligand is not induced as a result of damage response to UV, but isexposed as a result of the process of dying in response to certainstimuli.

CLEC9a Mediates an Adjuvant Signal Delivered by Dying Cells to DendriticCells.

UV-dead cells signal through the cytoplasmic domain of CLEC9A in a Sykand Y7-dependent fashion (FIG. 21). This system allowed us to explorewhether anti-CLEC9A antibodies could act as specific blocking reagentsfor this interaction and we found that both Fab and full antibodies insoluble form were powerful blocking reagents (FIG. 22). To test theeffects of dying cells in dendritic cells and their effector function invitro, we designed a cross-presentation assay in which UV-treated bm-1cells loaded with OVA protein were allowed to interact with Flt3L BMDCin the presence or absence of blocking anti-CLEC9A. Bm-1 cells are froma B6 haplotype but express a mutated H2K^(b) that does not bind theimmunodominant class I peptide for OVA (SIINFEKL; SEQ ID NO: 7). As areadout for DC capacity for cross-priming, specific OT-I cells wereadded to the assay. Proliferation of OT-I cells, as readout for theamount of antigen that was cross-presented, was not greatly affected(FIG. 18 a). However, cytokine production by OT-I cells, which isdependent in the help promoted by DC activated by an adjuvant effect,was severely inhibited when blocking CLEC9A antibodies were used (FIGS.18 a and b).

To confirm these results we generated mice deficient in CLEC9A,expressing EGFP under the control of Clec9a promoter (Clec9a^(egfp/−)).We generated Flt3L BMDC deficient or not in CLEC9A and we analyzedwhether the uptake of dying cells was affected. FIG. 18 c shows nodifference between in the capacity for uptake of dying cells betweenCLEC9A⁺ and CLEC9A⁻ Flt3L BMDC. Then, we assayed the effect of CLEC9Adeficiency in Flt3L BMDC in cross-presentation to OVA protein eitherloaded in UV-treated bm-1 cells or expressed intracellularly in anon-secreted OVA-GFP fusion protein in bm-1 MEFs that were UV treated(FIG. 18 d). OT-I proliferation was affected, suggesting a more profoundeffect in blockade of cross-presentation than the antibody blockade.However, at higher doses of OVA, including OVA expressed by bm1MEFs,proliferation was not affected and IFNγ production was severelyinhibited (FIGS. 18 d and e). These results suggest that there is ablockade in the adjuvant effect associated to dying cells duringcross-priming in vitro in the absence of CLEC9A in DCs.

CLEC9a Senses Immunogenic Cell Death to Promote Crosspriming In Vivo

As CD8α⁺ DC expressing CLEC9A are the main cell type characterized topromote cross-priming to dead cell associated antigen in vivo, we testedthe effect of CLEC9A blockade in this function. Mice that receivedUV-treated bm1 MEFs expressing OVA showed expanded CD8+ T cells againstOVA from the endogenous repertoire 6 days later (FIG. 19 a, left panel).These CD8+ T cells were able to produce IFN-γ in response to SIINFEKL,showing that they are effector cells and that dead cells behaved as animmunogenic carrier of antigen associated to adjuvant activity of deadcells (FIG. 19 a, right panel). Pre-treatment with anti-CLEC9A blockingantibody, but not with isotype control, blocked both the generation ofspecific CD8 response and its effector activity (FIG. 19 a).

To determine the precise role of CLEC9A in the process of immunogenicityof dead cells related to crosspriming to dead cell-associated antigen,we exploited the assay in CLEC9a^(egfp/−) mice. The results showed avery significant and partial inhibition of crosspriming to dead cellassociated antigen in vivo in the absence of CLEC9A (FIG. 19 b, leftpanel). As knock-out mice were generated in a mixed C57BL/6-129background and are being back-crossed with C57BL/6 (N3), we grouped the12 female litters whose individuals were pooled in FIG. 19 b, leftpanel, and we compared the average between CLEC9A+ and CLEC9A− mice,showing that 12 out of 12 litters showed reduction, with an average30.05% inhibition (p<0.0001, Student's t test) (FIG. 19 b, right panel).As shown in FIG. 19 b, right panel, differences in background penetranceamong different litters could explain significant variability incross-priming ability among litters and dampen the real differencebetween CLEC9A⁺ and CLEC9A⁻ mice, which is still very significant albeitonly partial. Moreover, IFN-γ production in response to SIINFEKL ex vivowas severely affected, showing the deficiency in effector responsegenerated via cross-priming to dead-cell associated antigen in theabsence of CLEC9A (FIG. 19 c). In conclusion, CLEC9A deficiency resultsin a reduced adjuvancy of the dead-cells with associated antigen thatleads to a blockade in specific T cell effector response to dead cellassociated antigen.

Methods

Mice

C57BL/6 mice, OT-I mice on a Rag−/− C57BL/6 background and B6.SJLbackground mice (congenic CD45.1°) were bred at Cancer Research UK inspecific pathogen-free conditions. K^(bm-1) mice were purchased from TheJackson laboratory (Bar Harbor, Me.; stock number 001060), and, togetherwith C57BL/6 mice, OT-I mice on a Rag−/− C57BL/6 background, MyD88-TRIFdouble knock out, Clec9a″, and Clec9a mice were bred at Cancer ResearchUK in specific pathogen-free conditions. Bone marrow chimeras were madefrom Syk-deficient fetal liver cells as previously described (Turner etal. Nature 378, 298 (1995)) All animal experiments were performed inaccordance with national and institutional guidelines for animal care.

Reagents

Culture medium was RPMI 1640 (Invitrogen) supplemented with glutamine,penicillin, streptomycin, 2-mercaptoethanol (all from Invitrogen) and10% heat-inactivated foetal calf serum (Bioclear). Antibodies used forflow cytometry analysis experiments were from BD Pharmingen and includedthose specific for CD11c (clone HL3, hamster IgG1), CD24, CD11b, B220,Ly6C, CD4 (RM4-5, rat IgG2a), CD8. Antibodies used for ELISA werecapture IFN-γ (XMG1.2, rat IgG1) detection/IL12-23 p40. Purified 2.4G2(anti-FcgRIII/II, rat IgG2b, used to block unspecific Ab binding) wasfrom Cancer Research UK antibody production service. For flow cytometry,cell suspensions were stained in ice-cold PBS supplemented with 2 mMEDTA, 1% FCS and 0.02% sodium azide. Data were acquired on a FACSCalibur(BD Biosciences) and analyzed using FlowJo software (Treestar, SanCarlos, Calif.).

Cells

Mouse bone marrow-derived DCs (BMDCs) were generated using GM-CSF andpurified from bulk cultures by magnetic selection with anti-CD1cmicrobeads (GM-CSF BMDCs). Alternatively, BMDCs were generated byculturing bone marrow cells in the presence of 100 ng/ml of Flt3L (R&D)for 10 days, by which time all living cells were positive for CD11c(Flt3L BMDCs). Spleen cells were prepared by liberase/DNAse digestionand enriched for DC by positive selection with anti-CD11c microbeads.OT-I T cells (from lymph nodes and spleen) were purified by negativeselection using a cocktail of biotinylated antibodies (anti-CD11c,CD11b, B220, FcγR, Gr-1, and CD4) followed by streptavidin microbeads.Human peripheral blood mononuclear cells (PBMCs) were prepared fromsingle donor leukocyte buffy coats (National Blood Transfusion Service)by sedimentation over Ficoll-Hypaque (GE-Healthcare).

PCR/RT-PCR

Total RNA was extracted using Trizol (Invitrogen) from subsets ofsplenic DC enriched in CD11c with anti-CD11c microbeads (Miltenyi) andsorted for CD4 and CD8 expression in a FacsAria sort (BD). In addition,RNA was extracted from sorted subsets of GMCSF and Flt3L in vitroderived BMDC. RNA was prepared by DNAse digestion (DNA-free, Ambion) andreverse transcribed using Superscript II reverse transcriptase (Gibco),1 μM dNTPs and 10 μM random hexanucleotides (Gibco). cDNA was amplifiedusing 35 PCR-cycles, consisting of 30 s 94° C., 30 s 55° C., 1 m 72° C.Sequences of primers were: mCLEC9a Fw 5′ AGACTGCTTCACCACTCCAA (SEQ IDNO: 9); mCLEC9a Rv: 5′ CTTGGCACAATGGACAAGGT (SEQ ID NO: 10; b-actin Fw:5′ GTTTGAGACCTTCAACACCCC (SEQ ID NO: 11), b-actin Rv: 5′GTGGCCATCTCCTGCTCGAAGTC (SEQ ID NO: 12); hClec9a Fw: 5′CCCAAGTCTCATTTGGAGGA (SEQ ID NO: 13; hClec9a-1 Rv: 5′AAATCTGGACGGTGTGGAAG (SEQ ID NO: 14).

Generation of anti-CLEC9a mAb

mAbs Against Murine Clec9a

Wistar rats were immunized with RBL-2H3 cells transfected with CLEC9afused to an HA epitope and fusion of splenocytes from hyper-immunizedrats with the rat myeloma cell line Y3 was performed following standardprocedures. For the detection of positives, we used the B3Z cell line²⁵expressing a chimera with the extracellular domain of CLEC9a, thetransmembrane region from NKRP1B and the intracellular tail of CD3ζ andfollowed by an IRES-GFP, a strategy that has been described²¹.

The cDNA sequence of the fusion chimera for mCLEC9a is shown in FIG. 3.The B3Z cell line contains a reporter for NFAT coupled to β-Gal activityand any engagement of the chimerical molecule results in the activationof NFAT and the reporter, that can be then revealed following standardassays for b-Gal activity. The screening for antibodies was byfunctional activation of the B3Z expressing the chimera compared withthe parental cell line. Those antibodies that were selected as positiveswere confirmed by FACS analysis in the parental cell line (EGFP−)compared to the CLEC9a chimera expressing cells (EGFP+). This methodallowed the selection of three mAbs named 1F6, 397, and 7H11 as shown inFIG. 4. mAb were then conjugated to biotin or to Alexa488 for staining(Invitrogen), or used for conjugation with S1 peptide.

mAbs Against Human Clec9a

BALB/c mice were immunized 3-4 times with RBL-2H3 cells expressing humanClec9a fused to an HA epitope. Fusion of splenocytes with the mousemyeloma line SP2/0 was carried out using standard procedures. Forhybridoma screening, we used the B3Z cell line, which expresses a β-galreporter for NFAT (23). This cell line was transduced with a retrovirusencoding a chimera of the extracellular domain of human Clec9a fused tothe transmembrane region from NKRP1B and the intracellular tail of CD3followed by an IRES sequence and the GFP gene (24). Hybridomasupernatants were screened for the ability to bind to the Clec9achimera, resulting in the activation of the NFAT reporter and inductionof β-gal activity (24). Supernatants that tested positive in this assaywere further screened by flow cytometry using a mixture of B3Z cellsexpressing the chimera Clec9a (GFP₊) and parental B3Z cells (GFP−). Thismethod allowed the selection of one mouse mAb specific for hClec9a (8F9(IgG2a)).

Flow Cytometry

Fluorochrome- or biotin-labeled antibodies specific for mouse CD11c,CD24, CD11b, B220, Ly6C, CD4 and CD8α were from BD Pharmingen. Purified2.4G2 (anti-FcγRIII/II) was from Cancer Research UK antibody productionservice. Mouse cell suspensions were incubated with 10 μg/ml of 2.4G2mAb to block Fcγ receptors and were then stained in ice cold PBSsupplemented with 2 mM EDTA, 1% FCS and 0.02% sodium azide. Forendocytosis studies, FcγR-blocked cells were labeled with 5 μg/ml ofbiotinylated anti-Clec9a mAb for 30 min at 4° C. Cells were then washedtwice and incubated for different times at 4° C. or 37° C. beforetransferring to ice and adding streptavidin PE. For in vivo labelingstudies, Alexa-488 conjugated anti-Clec9a or isotype-matched controlmAbs were injected i.v. at the indicated dose and tissues were preparedand analyzed after 16 h. Antibodies specific for human CD3, CD14, CD19,CD56, HLA-DR, CD34, CD123 and CD16 were purchased from BD, and CD1b/c,and BDCA-3 were from AbCam (Cambridge, UK). Human mononuclear cells wereblocked with 100 μg/ml human IgG (Sigma-Aldrich) and stained as above.Data were acquired on a FACSCalibur (BD Biosciences) and analyzed usingFlowJo software (Treestar, San Carlos, Calif.).

BM-DC Culture and Stimulation

GM-CSF BM-DC were generated as described²⁶ and DC were purified frombulk cultures with anti-CD11c microbeads before use (Miltenyi Biotec).BM-DC purity was checked by FACS and was routinely>98% (data not shown).Flt3L BMDC were generated from bone marrow cultured in the presence of75 ng/ml or 50 ng/ml of Flt3L (R&D) for 10 days. For cytokine productionand surface marker expression analyses, 5−10×10⁴ Flt3L BM-DC per wellwere cultured for 18-24 hours in 200 ml culture medium containing Flt3Lin 96-well flat-bottomed plates previously coated with isotype controlor anti-CLEC9a mAb. Cytokine levels in the supernatants were measured bysandwich ELISA using capture anti-IL-12 p40-p70 (C15.6) and detectionbiotin anti-IL12p40-p70 (C17.8), both from BD. For endocytosis in Flt3LBMDC, FcγR were blocked with 10 μg/ml of 2.4G2 mAb and cells were thencultured with 5 μg/ml of biotinylated mAb for 30 min at 4° C. or 37° C.,washed and allowed for 1.5 h or 0.5 h at the assay temperature beforewashing and adding simultaneously the secondary reagent (streptavidinPE) at 4° C. For confocal analysis, Alexa 488 conjugated mAb were addedto FcγR-blocked Flt3L-BMDC at 5 mg/ml for 30 min at 4° C. or 37° C. andthen washed and allowed for a further 1.5 h at the assay temperaturebefore allowing to adhere to poly-L-lysine-coated coverslips andfixation in 2% PFA for 20 min RT. Samples were mounted in slides withFluoromount (Southern Biotech, Birmingham, Ala.). A confocal series ofdifferential interference contrast and fluorescence images was obtainedsimultaneously with a laser scanning confocal microscope (Axioplan 2,Zeiss, Germany) with a 63°-Plan-Apochromat NA 1.4 oil objective. Imageanalysis was performed with LSM 510 software (Zeiss, Germany).

Peptide Pull-Downs and Western Blotting

For peptide pull-downs, biotin-conjugated peptides were dissolved in 40%DMSO before dilution in lysis buffer. Recombinant human Syk (Upstate)diluted in lysis buffer was incubated with the indicated biotinylatedpeptides corresponding to the CLEC9a and Dectin-1 intracellular tail(Cancer ResearchUK Peptide Synthesis Laboratory) andstreptavidin-sepharose (Sigma Biosciences AB, Uppsala, Sweden). Afteraffinity purification, Sepharose beads were washed once in lysis bufferand boiled in SDS gel-loading buffer containing 10% β-mercaptoethanol.Proteins were separated by sodium-dodecyl-sulfate-polyacrylamide gelelectrophoresis (SDS-PAGE), transferred onto Immobilon PVDF membranes(Millipore Corporation, Bedford, Mass.), and probed with rabbit anti-Syk(a combination of 2131 serum raised against a synthetic peptidecorresponding to amino acids 318-330 of murine Syk²⁰ and anti-Syk fromCell Signaling Technology, Inc., catalog number 2712, raised against asynthetic peptide corresponding to the C terminus of human Syk) followedby chemiluminescent detection.

In the LK cell activation assay, LK cells were plated in 6-well platescoated with anti-C9 or isotype control for the indicated times. Cellextracts were prepared in lysis buffer (50 mM HEPES [pH 7.4], 150 mMsodium chloride, 100 mM sodium fluoride, 10 mM tetrasodiumpyrophosphate, 1 mM sodium orthovanadate [pH 10.0], 1 mM EDTA [pH 8.0],1.5 mM magnesium chloride, 10% glycerol, 1% Triton X-100, 1 mM PMSF, and“Complete” protease inhibitor cocktail tablets [Roche]); insolublematerial was discarded and a fixed amount of lysate was run by SDS-PAGEas described above. For WB rabbit anti-Syk as above and anti-P-Syk,anti-Erk, anti-P-Erk were from Cell Signaling.

NFAT Reporter Assay in B3Z and BWZ Cells

B3Z cells containing a reporter plasmid for NFAT coupled to LacZactivity have been previously described²⁵. CLEC9a WT or a mutant versionY7F (Stratagene) were transduced in wt or Syk-transduced B3Z cells.Cells were plated in 96 well plates coated with isotype control oranti-CLEC9a mAb and after overnight culture, were washed in PBS andlysed in CPRG-containing buffer as described²¹. Four hours later A595was measured, using OD 655 as a reference.

For detection of CLEC9A ligands, BWZ cell line was transduced with aretrovirus encoding a chimera of the extracellular domain of mouse orhuman CLEC9A fused to the transmembrane region from NKRP1B and theintracellular tail of CD3ζ followed by an IRES sequence and the GFP gene(24). Ligand binding to the CLEC9A chimera would result in theactivation of the NFAT reporter and induction of β-gal activity. Toassay basal activation of BWZ cells expressing mouse and humanCLEC9A-CD3ζ chimeric receptors, 4, 2, 1, or 0.5×10⁶ cells/ml werecultured in 3 ml in a 6 well plate and allowed to (over) grow for twodays. Frequency of dead cells was determined using TO-PRO3 dye(Invitrogen) and live cells were plated in fresh medium at 2×10⁵/well in96-well flat bottom plates. After overnight culture, LacZ activity wasmeasured as above.

To determine exposure of ligand in different cell types, LK (B cellline), SV-40 immortalized mouse embryonic fibroblasts (MEFs) derivedfrom bm-1 mice {Ref} using standard protocols, RBL rat leukemia, 3T3fibroblasts, and HEK-293 human embryonic kidney cells were UV irradiated(240 mJ/cm²) and left 24 h to induce cell death before adding to 2×10⁵BWZ transfectants in a 96-well flat bottom plate at a 1:1 ratio in thepresence or absence of control Fab or anti-mouse CLEC9A (1F6) andanti-human CLEC9A (8F9) Fab (Sancho et al, J Clin Invest. 2008;118(6):2098-110). After overnight culture, LacZ activity was measured asabove. In some assays, BWZ cells transfected with CLEC9A wt were used todetermine if the ligand signals through the cytoplasmic tail andevaluate full antibodies as blocking reagents comparable to the Fab.

UV dose response was performed irradiating LK cells with the followingdoses of UV (mJ/cm²): 0, 0.5, 1.5, 5, 15, 50, 240. Frequency of deadcells was determined 24 h later using to-pro3 dye and cells were plated1:1 with BWZ transfectants (2×10⁵ cells/well) in fresh medium. Afterovernight culture, LacZ activity was measured as above.

Different treatments for LK cells and bm-1 immortalized MEFs wereevaluated in exposure of the ligand. Cells were cultured for 24 h withMitoxantrone (1 μM) or in the absence of serum (serum deprivation).Osmotic shock was performed as previously described (Liu et al. 2002. J.Exp. Med. 196: 1091-1097). Three cycles of freeze-thaw were performed inthe pellet of cells, freezing in liquid N₂ and thawing at 37° C. Afterthese treatments, Frequency of dead cells was determined using to-pro3dye and cells were stained for rsCTLD as indicated below. Cells wereplated 1:1 with BWZ transfectants (2×10⁵ cells/well) in fresh medium.After overnight culture, LacZ activity was measured as above.

Coupling of SIINFEKLC (SEQ ID NO: 6)-biot (S1) to mAb.

To conjugate the immunodominant OVA peptide SIINFEKL (SEQ ID NO: 7) tothe bivalent antibody, a derivative of the SIINFEKL (SEQ ID NO: 7)peptide, named 51, containing an added Cysteine (C) to generate a freesulfhydril group and biotin to track the labelling was synthesized andpurified by high-performance liquid chromatography at Cancer ResearchUK. The mAb were treated with sulfo-SMCC 30 min at RT, generatingsulfo-reactive groups in the tertiary amines, followed by purificationof the activated antibody in a molecular size exclusion chromatographycolumn (Pierce). Then, the 51 peptide was freshly prepared and allowedto react in equimolecular amounts with the activated antibody 1 h at 37°C., and purified in a chromatography column. The extent of biotinylationof the mAb allowed us to quantify in 1-1.2 peptides coupled per mAbmolecule in all antibody conjugates generated, using the Fluoreporterkit (Invitrogen) and following manufacturer's instructions.

Coupling of S2 Peptide and Melanocyte Differentiation Antigen Peptidesto mAb

S2 peptide (SIINFEKLTEWTSSNVMEERC-biotin) was synthesized and purifiedby HPLC at Cancer Research UK. mAbs in PBS were treated with sulfo-SMCCfor 30 min at room temperature to generate sulfo-reactive groups intertiary amines. The activated antibody was purified by molecular sizeexclusion chromatography (Pierce), S2 peptide was added (2:1 molarratio) and the reaction was allowed to proceed for 1 h at 37° C.Conjugates were purified using an Immunobind sepharose column. Theextent of biotinylation of the mAb was assessed using the Fluoreporterkit (Invitrogen) as per manufacturer's instructions.

The same strategy was used to synthesize peptides from the melanocytedifferentiation antigens, gp100 (EGSRNQDWL (SEQ ID NO: 15) and KVPRNQDWL(SEQ ID NO: 16); H-2 Db-restricted (25)), TRP-1 (TWHRYHLL (SEQ ID NO:17) and TAYRYHLL (SEQ ID NO18); H-2 Kb-restricted (26)) and TRP-2(SVYDFFVWL SEQ ID NO: 19); H-2 Kb-restricted (27)), each modified byaddition of cysteine-eahx-biotin at the C-terminus.

Targeting In Vivo with Anti-CLEC9a mAb.

Anti-CLEC9a, isotype control, or anti-DEC-205 were coupled to Alexa488(Invitrogen) or to S1 peptide. For the experiment of targeting usingAlexa488 Abs, mAb-Alexa488 were injected i.v. (20 μg) and splenocyteswere extracted and analyzed after 16 h.

In the experiment of targeting of antigen to specific subsets in vivo,S1-Abs were injected i.v. (5 μg) and splenocytes were extracted andpurified in the CD11c positive and negative fraction with anti-CD11cmicrobeads (Miltenyi Biotec). OT-I cells were obtained from lymph nodesand splenocytes of OT-I Rag−/− mice and purified by negative selectionusing a cocktail of biotinylated antibodies (anti-CD11c, CD11b, B220,FcgR, Gr-1, and CD4) followed by streptavidin microbeads (MiltenyiBiotec). Different amounts of in vivo targeted DC, as indicated in theFigure legend, were cultured with 10⁵ OT-I cells labelled with 2 μM CFSE(Invitrogen) in U-bottomed plates. Three days later, proliferation wasdetermined by CFSE dilution in cells positive for Vβ5.1 and CD8 andnegative for TO-PRO 3. Cells were acquired with true count beads ( . . .) to quantify the absolute number of cells. IFN-γ in the supernatantswas determined by sandwich ELISA.

In the experiment of targeting in vivo to evaluate specific CTLresponse, mAb-S1 were injected s.c. in the hind paws (2 μg) together ornot with 25 μg of anti-CD40 (3/23, BD Pharmingen) and five days later invivo killing assays were performed as described'. Briefly, targetsplenocytes from B6.5JL background (congenic CD45.1⁺) were loaded with20 nM, 200 nM or no SIINFEKL (SEQ ID NO: 7) peptide and respectivelylabelled with 0.03 μM, 0.3 μM or 3 μM of CFSE for 20 min at 37° C.Labelled splenocytes (10⁷) were i.v. injected. The following daysplenocytes were extracted and the CD45.1 positive population wasanalyzed for CFSE. In addition, 5×10⁵ splenocytes were cultured in thepresence or absence of 1 μM SIINFEKL (SEQ ID NO: 7) for 24 h andsupernatants were quantified for IFNγ by ELISA. Blood and spleen cellswere also labeled with SIINFEKL(SEQ ID NO: 7)-H2 Kb tetramer (BeckmanCoulter), anti-CD8 and anti-Thy 1.2 and analyzed for the % tetramer+cells among the CD8+ T cell population.

B16 Melanoma Tumor Therapy Model.

B16 melanoma cells were transduced with OVA-GFP fusion protein andsorted for GFP expression. Tumor cells (2×10⁵) were injected i.v. in thetail of congenic B6 mice and 6 days later the therapeutic treatmentconsisting of Ab-S1+anti-CD40 was injected s.c. in the paw. At day 18post-tumor injection, lung tumors were counted. Tumor therapyexperiments were done in an analogous fashion except that mice receivedB16-OVA 3 days prior to antibody treatment.

Tumor therapy and prophylaxis experiments were also carried out withnon-transduced parental B16 cells. These were given i.v. (5×105/mouse)either 3 days before (therapy) or 1 day after (prophylaxis) immunizationwith anti-CLEC-9a or control antibody covalently coupled to a mixture of5 peptides derived from gp100, TRP-1 and TRP-2 (1 μg/paw) together withanti-CD40 (12.5 μg/paw) and poly 1:0 (5 μg/paw). Tumor burden wasassessed by counting lung foci. When these were too numerous to count(>250 per mouse), they are shown as 250. CTL responses were monitored asdescribed above.

Generation of Clec9a^(−/−) Mice.

Mice were generated using Red/ET recombineering (Gene Bridges,Heidelberg, Germany) to capture directly the region of the gene tomodify from the BAC clone RP-23 248-K14 (C57BL/6 BAC clone fromInvitrogen). A conventional gene-targeting replacement vector:pFloxRI+tk, which uses the strategy of both positive (neomycinresistance gene) and negative (herpes simplex virus thymidine kinasegene) selection for the isolation of homologous recombinant ES cellsclones was amplified by PCR using the primers indicated with 20nucleotides pairing with the vector and 70 nucleotides pairing withregions of Clec9a to capture. The primers used were Fw 3arm 24330 pFlox5′ ATAATATCAT ATTTCTATAA TATCATTGTA ATGACAAAAC CACTGAACTA GTGCCTGTAAAGGCAGGAGG GGTACCGAGC TCGAATTCTA CCG 3′ SEQ ID NO: 20); Rv pFlox 5arm5′TGCTATATTA CAGATTTTCA AGTGGGGTAG CCTGGAGTAA CAAGATGGCA GGGCATAATCACTAGTGCGG CCGCCACCGC GGTGGAGCTC CAGCTTT (SEQ ID NO: 21) 3′.

Once the region to modify was included in the Amp-resistant vector, acassette including farnesylated EGFP, and the PGK-gb2 promoter followedby Kan/Neo allowed to repeat the recombineering homologous recombinationstep with selection for Kan. The primers used for amplification of theEGFP-F Kan-Neo resistant cassette were: Rv NeoKan 3arm 5′ TGCTTTTGTACTTACACTTG ATGCCCAAGA AAATGGACGT TGCTAACAAG CCCATACAGA CCACACCTCGAGATAACTTC GTATAATGTA T3′; (SEQ ID NO 22) and Fw 5arm EGFP-F 5′TTTGTGCCAG GCTCCTATGT AGACTGCTTC ACCACTCCAA GCGCCTTCAG CATGCATGTCGACATGGTGA GCAAGGGCGA GGAG 3′(SEQ ID NO: 23).

The targeting vector was prepared to express EGFP-F with a strong polyAimmediately downstream the first two amino acids from CLEC9A anddisrupted exons 1 and 2, terminating transcription with the strong polyAThe targeting vector was linearized prior to transfection using Not I.Transfection of S6B6 hybrid 129S6/C57BL/6 F1 derived embryonic stem (ES)cells was achieved by electroporation and recombinant clones wereisolated after culture in G418 and gancyclovir. ES cells survivingselection were screened by PCR using two independent primer pairs withone of the primers external to the short arm. The primer pairs usedwere: Scr Fw1 5′ GATCTGTGTG TTGGTTTTTG TGTGC 3′(SEQ ID NO: 24); Scr Rv1,5′ TAGCATGGCA CTTCTCCATT ACCTT 3′ (SEQ ID NO: 25) Amplicon Fw1Rv1: 2138bp. Scr Fw2, 5′ GCGAATTCGG TACCAATAAA AGAGC 3′ (SEQ ID NO: 26); Scr Rv2,5′ CAGAAGCTTC CTGGTTTTGG TTTTT 3′ (SEQ ID NO: 27) Amplicon Fw2Rv2: 2352.

Correctly targeted, karyotypically euploid ES clones were micro-injectedinto 3.5 day post coitum C57BL/6 blastocysts and resulting offspringwith coat-color chimerism were bred with C57BL/6 females to identifygerm-line transmission. Germ-line transmitting chimeras weresubsequently bred with C57BL/6 females to secure the gene-targetedallele in the pure C57BL/6 background. Heterozygous animals wereinterbred to generate homozygous deficient animal and matched littermatecontrols. The expression of NK1.1 C57BL/6 gene, ligated to Clec9a, inthe knock out mice shows that the homologous recombination of thetargeted C57BL/6 BAC clone was integrated in the C57BL/6 copy of the F1in S6B6 ES cells.

Clec9a−/− mice used in this work were on a mixed 129/Sv x C57BL/6genetic background in the third generation of backcrossing to C57BL/6.

Recombinant Soluble CTLD Generation.

The CTLDs for mouse CLEC9A and Dectin-1 were independently cloned inframe in the p3xFlag-CMV-9 expression vector from Sigma with an addedBirA monobiotinilation sequence. Primers used for CTLD amplificationwere mCLEC9A Fw 5′GGATCC mCLEC9A Rv 5′GGATCC mDectin Fw 5′GGATCC3′mDectin Rv 5′GGATCC3′ CHO cells were transfected and selected with G418(1 mg/ml). Stable transfectants were cloned twice by limiting dilution,selecting clones secreting rsCTLD from Dectin-1 or CLEC9A to thesupernatant, detected by a sandwich ELISA using as capture anti-flag M2(Sigma) and for detection biotin-2A11 anti-Dectin-1 or biotin-1F6anti-CLEC9A. Concentrated supernatants from CHO clones were generated inCELLine bioreactors (Integra Biosciences, Chur, Switzerland) and werepurified using anti-flag M2 agarose (Sigma). Monobiotinylation was thenperformed using standard procedures {Ref} and tetramers were generatedusing PE-Streptavidin (Sigma). PE-tetramers of rsCTLD were then used forstaining of dead cells or zymosan for 30 min at 4° C. in normal FACSbuffer. Samples were counter-stained with to-pro3 and acquired by flowcytometry.

Cross-Presentation In Vitro.

In the cross-presentation assay in vitro we co-cultured three celltypes: dead cells loaded with OVA or expressing OVA, Flt3L BMDC fromdifferent origins in the presence or absence of antibodies blockingCLEC9A and the readout, OT-I OVA-specific transgenic T cells. We testedFlt3L BMDC from CLEC9a^(−/−) or CLEC9a⁺ littermates. In addition, Flt3LBMDC from C57BL/6 were cultured in the absence or presence of controlFab, or anti-CLEC9A (1F6) Fab (10 μg/ml), as well as with fullantibodies against CLEC9A or isotype control (20 μg/ml). Whereindicated, CD8α-like Flt3L BMDC with CD11b^(lo) and CD24^(hi), asdescribed (19), were sorted. As a source of dead cells associated to OVAantigen for cross-presentation, we used bm-1 splenocytes, a C57BL/6congenic mouse that express a mutation in the H2K^(b) molecule thatprevents the binding of SIINFEKL, the immunodominant class I OVA peptidefor H2K^(b). In that way, cells loaded with OVA will not be able topresent directly the OVA peptide and should be processed andcross-presented to generate a response. To load the OVA by adsorption,we incubated the indicated doses of soluble OVA (Calbiochem) with lowlevels of endotoxin with bm-1 splenocytes in PBS for 20 min beforewashing five times in PBS. Alternatively, we generated bm-1 mouseembryonic fibroblasts (MEFs) and we immortalized them with SV-40 T largeantigen. Then, we transduced then with a truncated non-secreted OVA-GFPfusion protein and sorted for homogeneous expression of OVA-GFP. Both,OVA-loaded splenocytes and OVA-expressing MEFs were then UV-irradiated(240 mJ/cm²) and cultured overnight in complete medium. The followingday, splenocytes were co-cultured 5:1 ratio to Flt3L BMDC (10⁵cells/well, 96 U-bottom) and OVA-MEFs were cultured with Flt3L BMDC in a1:1 ratio. OT-I cells negatively selected (purity>80%) using MACS beads(Miltenyi) and labelled with CFSE (2 μM) were added to the assay (10⁵cells/well). Three days later supernatants were harvested to detect IL-2and IFN-γ by sandwich ELISA and cells were stimulated with PMA (10ng/ml) and Ionomycin (500 ng/ml) for 4 h, adding Brefeldin A (10 μg/ml,Sigma) during the last 3 h of culture. Cells were then stained for Vβ5,CD8 and IFN-γ by intracellular staining and acquired by flow cytometryto determine absolute counts, loading samples with a fixed number ofcalibration beads (BD) and IFN-γ production by intracellular staining.

Uptake of Dead Cells

WT or CLEC9a^(−/−) CD8α-like Flt3L BMDC were incubated for 2 h withdifferent ratios of splenocytes that were treated 24 h before with UVC(240 mJ/cm²) and labelled with PKH26 (Sigma). As Flt3L BMDC werelabelled for CD24, double positive for PKH26 and CD24 could be due tobinding (4° C.) or binding+ uptake (37° C.) of dying cells and thefrequencies were quantified by flow cytometry for each type of DC.

Cross-Presentation In Vivo

OVA-expressing immortalized bm-1 MEFs generated as indicated above wereUVC-treated (240 mJ/cm²) and cultured overnight before being injectedi.v. (0.75×10⁶ cells/mouse). Mice (C57BL/6) were pre-treated with ani.p. injection of PBS or 400 μg isotype control (AFRO-MAC 49, rat IgG1)or 1F6 anti-CLEC9A 30 min before the i.v. injection. Alternatively,Clec9a^(−/−) or littermates Clec9a⁺ were used. Six days later, inductionof CD8′ T cell effector response arising from the endogenous repertoirewas tracked by the number of H2K_(b)-OVA peptide tetramer positive cellsand IFNγ production in response to SIINFEKL (SEQ ID NO: 7) ex vivo inthe CD8 subset. In FIGS. 19 c and 19 d in which data were pooled fromseveral litters in independent experiments, we performed a normalizationof the data before pooling. Each litter (females) was considered anindependent experiment and raw data in each litter were normalized tothe mean of Clec9a+ mice in that litter and multiplied by the arithmeticmean obtained for the total population of Clec9a+ mice in all thelitters in independent experiments. For the analysis of fold change inlitters, arithmetic mean for Clec9a+ mice or Clec9a^(−/−) mice wascalculated in each litter. All 12 litters out of 12 analyzed showed foldreduction in the arithmetic mean of % tetramer positive cells in CD8 Tcell subset of Clec9a^(−/−) mice compared to Clec9a+.

Statistics

Statistical analysis was performed with a two-tailed Student's t testfor differences among groups or U Mann-Whitney when normality of datacould not be inferred. p<0.05 was considered statistically significant.Quantitative data are expressed as means±SEM unless otherwise stated.

While the invention has been described in conjunction with the exemplaryembodiments described above, many equivalent modifications andvariations will be apparent to those skilled in the art when given thisdisclosure. Accordingly, the exemplary embodiments of the invention setforth are considered to be illustrative and not limiting. Variouschanges to the described embodiments may be made without departing fromthe spirit and scope of the invention. All documents cited herein areexpressly incorporated by reference.

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The invention claimed is:
 1. A method of stimulating an immune responseagainst a peptide antigen in a subject, comprising administering to thesubject a composition comprising the antigen, wherein the antigen isassociated with an antibody or functional fragment thereof havingspecific affinity for a CLEC9a protein of SEQ ID NO: 2 or SEQ ID NO: 4,and an adjuvant; wherein said antigen is obtained from a parasite, apathogen or a cancer cell, and wherein said response includes T cellproliferation.
 2. A method according to claim 1 comprising more than oneadministration of said composition.
 3. A method according to claim 1wherein the immune response to be stimulated is a CTL response.